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The structure-function relationship of glucose-dependent insulinotropic polypeptide Morrow, Glenn Wesley 1994

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THE STRUCTURE-FUNCTION RELATIONSHIP OF GLUCOSE-DEPENDENT INSULINOTROPIC POLYPEPTIDEByGLENN WESLEY MORROWB.Sc., The University of British Columbia, 1989A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYnTHE FACULTY OF GRADUATE STUDIESDepartment of PhysiologyWe accept this thesis as coifonningto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAjune. 1994©Glenn Wesley Morrow, 1994In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)______________________________Department of /-/ V /0 Lo1 YThe University of British ColumbiaVancouver, CanadaDate______________DE-6 (2/88)ABSTRACTGlucose-dependent insulinotropic polypeptide or gastric inhibitory polypeptide(GIP) is a 42 amino acid endocrine gut hormone which exhibits several direct andindirect effects on fat and glucose metabolism. The first known, and mostscrutinized, metabolic function of the hormone was the potentiation of insulinrelease from pancreatic 13-cells in the presence of threshold glucose levels. Inorder to determine the region(s) of the molecule involved in mediating the insulinresponse at the beta cell, synthetic and proteolytic fragments of the molecule weregenerated and tested for their ability to potentiate the insulin response in theisolated, perfused rat pancreas. Previous work by others suggested that GIP aminoacids 15 to 30 might be necessaly for biological activity yet a synthetic 15-30fragment was biologically-inactive. However, enterokinase treatment of asynthetic 15 to 30 fragment restored approximately 30% of the integratedinsulinotropic activity over a 25 mm perfusion of the isolated rat pancreas. Thehypothesis that the restoration of biological activity was due to the enzymaticremoval of the amino terminus aspartic acid/lysine residues of G1P1530 wassupported by the observation that a synthetic fragment lacking these two residueswas also insulinotropic. There was no apparent difference in the insulin responsesto the synthetic fragment or the enterokinase-derived fragment. Furtherfractionation of the molecule generated a 19-30 fragment which was alsobiologically active suggesting that the residues necessaiy for the insulin responsewere contained within this region. Two recombinant prokaiyotic expressionsystems for GIP were developed to further define the bioactive residues across the1119-30 region and to establish a system for the generation of large amounts ofbiologically-active material. Site-directed mutagenesis of a recombinant GIP clonewas used to generate a full-length GIP molecule with alanine residues substitutedacross the 23 to 26 region of the polypeptide. This substitution resulted in the lossof biological activity suggesting that one or more of these residues is critical to theinsulin response. An innate intramolecular interaction associated with theexpression system precluded the isolation of sufficient quantities of alaninesubstituted material in the 19-22 and 27-30 regions of the molecule.111TABLE OF CONTENTSPageABSTRACT iiLIST OF TABLES xiLIST OF FIGURES xiiACKNOWLEDGMENTS xviiINTRODUCTION 1GIP; historical aspects 3GIP3.42 6Direct and indirect metabolic effects of GIP 7GIP isoforms 8Purification of native GIP 8The GIP receptor 11The insulinotropic domain of GIP 13Recombinant GIP 18Site-directed mutagenesis of cloned DNA 20Alanine scanning mutagenesis of GIP 23Synopsis 27ivMETHODSPageI. Purification of native porcine GIP 29II. Enterokinase digest of porcine G1P15..30 30III. Tryptic digest of porcine G1P1530 30IV. Construction of pRIT 2T hGIP, pRIT 5 hGIP 30V. PRIT 2T hGIP; expression of the protein A- 35hGIP fusion protein in E. co/iVI. Purification of recombinant hGIP 361) Cell lysate; supernatant 362) Cell lysate; insoluble debris 38VII. PRIT 5 hGIP; expression of the protein A- 38hGIP fusion protein in F. co/iVIII. Construction of pGEX 2T hGIP 39IX. Polymerase chain reaction mutagenesis of 42the human GIP cloneX. Expression of the mutagenic hGIP/GST fusion 45protein(s) in F. co/iXI. Purification of the recombinant hGIP/GST 46fusion proteinsXII. Optimization of GST/hGIP fusion protein 46expressionXIII. Cyanogen bromide cleavage of the GST/hGIP 48fusion proteinsVpageXIV. Purification of hGIPLeU and hGIPLeU 14 48alanine-substituted recombinant proteins1) Purification of hGIPLeu 4 482a) Purification of hGIPLeu 14, Ala 23-26’ protocol 1 492b) Purification of hGIPLeU 14, Ala 23-26’ protocol 2 492b. 1) Thrombin cleavage of GSThGIPLeu 14, Ala 23-26 492b.2) Purification of the GSThGIPLeu 14, Ala 23-26 50digestion products2b.3) CNBr cleavage and HPLC purification of 50gsm-hGIP 14, Ala 23-263a) Purification of hGIPLeu 14, Ala 19-22 aild 50hGIPLeu 14, Ala 27-30’ protocol 13b) Purification Of hGIPLeu 14, Ala 19-22 and 51hGIPLeu 14. Ala 27-30’ protocol 2XV. Sodium dodecyl suiphate-polyacrylamide gel 51electrophoresisXVI. Enzyme-linked immunosorbent assays (ELISA) 521) Sensitivity of anti-GIP(7-5-7) to hGIP 52and porcine GIP2) Confirmation of hGIP immunoreactivity 53by ELISAXVII. Western blot analysis 53XVIII. Amino acid sequence analysis of recombinant proteins 54XIX. Pancreas perfusion bioassay for GIP 54XX. Expression of results 55viRESULTSPageGIP fragment generation 56a) Enterokinase digest of synthetic porcine 56G1P15..30b) Tryptic digest of synthetic porcine G1P1530; 56purification and sequence analysis of theproductII. Analysis of the GIP fragments 59a) Pancreas perfusion bioassay of the 59enterokinase digestion productsb) Pancreas perfusion bioassay of 59synthetic GIP17-30c) Pancreas perfusion bioassay of 63G1P1930III. Prokaryotic expression of human GIP as a protein A 63fusion proteina) Expression of human GIP as a Staphylococcus 63aureus protein A fusion protein in Escherichiacoilb) Enzymatic cleavage of the protein A fusion 67protein with thrombinc) ELTSA analysis of recombinant hGIP 69d) Amino acid sequence analysis of recombinant 69GIPviiPagee) Bioassay of recombinant hGIP 73IV. Prokaryotic expression of human GIP as a glutathione 73S-tansferase fusion proteina) Expression of human GIP as a Schistosoma 73japonicurn glutathione S-transferase fusionprotein in Escherichia coilb) Optimization of GSTGIPLeu 4 expression 761) Expression of GSTGIPLeu 14 as a 76function of bacterial shain2) Induction time of gene expression versus 76fusion protein yieldsc) CNBr cleavage of the GSTGIPLeu 14 fusion 76proteind) Purification of hGIPLeU 14 from the CNBr 79digestion mixturee) Amino acid sequence analysis of recombinant 84hGIPLeu 14f) Bioassay of recombinant hGIPLeU 14 84V. Alanine scanning mutagenesis of hGIPLeU 14 84a) Purification of GST-hGIPLCU 14. Ala 23-26 from 87the transformed E. co/i topp 3 cell lysateb) CNBr cleavage of the GSThGIPLeu 14, Ala 23-26 87fusion proteinc) Purification of hGIPLeu 14, Ala 23-26 following the 87CNBr digestionviiiPaged) Amino acid sequence analysis of recombinant 90of hGIPLeU 14, Ala 23-26e) Bioassay Of hGIPLeu 14, Ala 23-26 90f) Purification of GSThGIPLeu 14, Ala 19-22 from 90the transformed E. co/i topp 3 cell lysateg) CNBr cleavage of the GSThGIPLeu 14. Ala 19-22 95fusion proteinh) Purification of GSThGIPLeu 14. Ala 27-30 fr0m 98the transformed E. co/i topp 3 cell lysatei) CNBr cleavage of the GSThGIPLeu 14, Ala 27-30 98fusion proteinVI. Alternative cleavage and purification strategy for 102GSThGIPLeu 14. Ala 19-22 and GSThGIPLeu 14, Ala 27-30a) Thrombin digestion of GSThGIPLeu 14, Ala 19-22 102GSThGIPLeu 14, Ala 23-26 andGSThGIPLe1114. Ala 27-30b) HPLC analysis of thrombin-digested 104GSThGIPLeu 14. Ala 19-22’ GSLhGIPLeu 14, Ala 23-26and GSThGIPLeu 14, Ala 27-30c) Optimization of the thrombin cleavage 104reactiond) Cyanogen bromide digestion of gsm- 107hGIPLeu 14. Ala 19-22’ gsm-hGIP 14, Ala 23-26and gsm-hGlP1114. Ala 27-30e) Reverse phase HPLC purification of cyanogen 107bromide-treated gsm-hGIP 14, Ala 23-26ixPage1) Amino acid sequence analysis of 107recombinant hGIPLCU 14, Ala 23-26peak 22) Bioassay of recombinant 110hGIPLeu 14, Ala 23-26 peak 2f) Reverse phase purification of cyanogen 110bromide-treated gsm-hGIP 14, Ala 19-22 andhGIPLeu 14, Ala 27-30DISCUSSIONI.. Fragment analysis of porcine GIP 114II. Prokaiyotic expression of GIP 118a) Expression of human GIP as a protein A 118fusion proteinb) Expression of human GIP as a glutathione 121S-transferase fusion proteinIII. Site directed mutagenesis of hGIPLCU 14 123IV. Cleavage and analysis of the GST-hGIPLCU 14 mutants 123V. Cleavage of the recombinant GIP fusion proteins: an 130overviewVI. Synopsis: the insulinotropic domain of GIP 133FUTURE STUDIES 136APPENDIX 1; LIST OF ABBREVIATIONS 138REFERENCES 140xLIST OF TABLESNumber Title Page1 Amino acid sequence homologies in the glucagon 24family of peptides2 F. coil culture densities at harvest 473 Result of N-terminal sequence analysis of recombinant GIP 724 Predicted proteolytic fragments generated following the 81exposure of GSThGIPLeu 14 to cyanogen bromide5 Digestion conditions for the cleavage of gsm- 106hGIPLeu 14, Ala 19-22 and gsm-hGIP 14. Ala 27-306 Sumiriary of experimental data 113xiLIST OF FIGURESNumber Title Page1 GIP sequence homologies among species 92 Fragment analysis of the GIP molecule 163 Construction strategy for the generation of pRIT 2T 32hGIP and pRIT 5 hGIP4 AcL-fibrinogenlhGlP construct 345 Purification strategy for protein A-hGIP 376 Construction strategy for the generation of hGIPLeu 14 407 Oligonucleotides used in the construction of hGIPLeU 14 418 PCR mutagenesis strategy of pUC 19 hGIPLCU 14 439 PCR mutagenesis primers used in the generation of 44the alanine mutants10 Cleavage sites in the 15 to 30 domain of GIP 5711 Reverse phase HPLC profiles resulting from the 58enterokinase digest of GIP153012 Reverse phase HPLC profiles resulting from the 60tiypsin digest of G1P15..30xiiNumber Title Page13 Effect of a linear gradient of enterokinase-derived 61peak 3 on the insulin response of the isolatedperfused rat pancreas14 Effect of a linear gradient of synthetic G1P17.30 62on the insulin response of the isolated peifusedrat pancreas15 A comparison of the insulin responses of the isolated 64perfused rat pancreas to enterokinase peak 3 andsynthetic porcine G1P173016 Effect of a linear gradient of G1P1930 on the insulin 65response of the isolated perfused rat pancreas17 SDS-PAGE analysis of the expression and purification 66of hGIP from the pRIT 2T expression system18 SDS-PAGE analysis of the SDS requirements for 68the thrombin digestion of protein A-hGIP19 Reverse phase HPLC profiles of porcine and 70recombinant human GIP20 ELISA analysis of recombinant hGIP 7121 Effect of a linear gradient of recombinant human 74GIP or porcine GIP on the insulin response of theisolated perfused rat pancreas22 SDS-PAGE comparison of the induced expression 75of glutathione S-transferase and glutathione Stransferase/hGIPLeU 14 fusion proteinsxiiiNumber Title Page23 SDS-PAGE and western blot analysis of the influence of 77bacterial strain on GSThGIPLeu 14 expression levels24 SDS-PAGE and western blot analysis of the influence of 78induction time on GSThGIPLeu j4 expression levels25 SDS-PAGE and western blot analysis of the CNBr cleavage 80and purification of hGIPLeU 14 from theGSThGIPLeu j fusion protein26 Reverse phase HPLC profile of CNBr-digested 82GSLhGIPLeu 1427 HPLC suiphapropyl profile of partially purified 83hGIPLeu 1428 Reverse phase HPLC profile of purified hGIPLeu 14 8529 Effect of a linear gradient of recombinant human 86GIPLeu 14 or porcine GIP on the insulin response ofthe isolated perfused rat pancreas30 SDS-PAGE and western blot analysis of the CNBr cleavage 88and purification of hGIPLeU 14. Ala 23-26 from theGST-fusion protein31 Reverse phase HPLC profile of CNBr-digested 89GSThGIPLeu 14. Ala 23-2632 HPLC sulphapropyl profile of partially purified 91hGIPLeu 14, Ala 23-2633 Reverse phase HPLC profile of purified hGIPLeU 14, Ala 23-26 92xivNumber Title Page34 Effect of a linear gradient of recombinant human 93GIPLeu 14, Ala 23-26 or porcine GIP on the insulinresponse of the isolated perfused rat pancreas35 SDS-PAGE and western blot analysis of the purification 94of GSThGTPLeu 14, Ala 19-2236 SDS-PAGE and western blot analysis of the CNBr 96cleavage of hGIPLeU 14, Ala 19-2237 Reverse phase HPLC profile of CNBr-digested 97GSLhGIPLeu 14, Ala 19-2238 SDS-PAGE and western blot analysis of the purification 99of GSThGIPLCU 14, Ala 27-3039 SDS-PAGE and western blot analysis of the CNBr 100cleavage of GSThGIPLeu 14, Ala 27-3040 Reverse phase HPLC profile of CNBr-digested 101GSThGTPLe1114, Ala 27-3041 SDS-PAGE analysis of the thrombin-digested, 103alanine-substituted mutants of GIP42 Reverse phase HPLC analysis of the thrombin- 105digested, alanine-substituted recombinant GIPfusion proteins43 Reverse phase HPLC analysis of CNBr-digested 108gsm-hGIP 14, Ala 23-2644 SDS-PAGE and western blot analysis of CNBr-digested 109gsm-hGIP 14, Ala 23-26xvNumber Title Page45 Effect of a linear gradient of recombinant human 111GIPLeu 14, Ala 23-26 peak 2 or porcine GIP on the insulinresponse of the isolated perfused rat pancreas46 Reverse phase HPLC analysis of CNBr-digested 112gsm-hGIP 14, Ala 19-22 and gsm-hGIP 14, Ala 27-3047 Modification strategy of the pGEX 2T-hGIP clone 13248 Amino acid sequence homologies at the carboxy 134terminus of selected glucagon family membersxviACKNOWLEDGMENTSI would like to take this opportunity to thank several people who made asignificant contribution towards the completion of this work. My supervisors, Dr.John Brown and Dr. Ross MacGillivray, for their patience and guidance as well asfinancial and moral support. Dr. Ray Pederson and Leslie Checknita, for theirdirect contribution to this work by peiforming the biological assays as well as Dr.Pederson’s guidance in the preparation of this manuscript. Dr. Billy Chow, whotaught me the fundamentals of molecular biology and who guided me throughmuch of the early work. Dr. Chris Mcintosh, who was always available to discussall things scientific and who provided a dissenting and rational voice when Ithought I had discovered something truly earth-shattering. I would like to thankTim Kieffer who quickly learned that he was never a match for me “one on one.”I would like to express my particular appreciation to my wife Sandie withoutwhose patience and forbearance I would still likely still be writing the introduction(and probably as a single man). A final note of gratitude to my parents, Edith andWesley Morrow, who always believed even when I wasn’t so sure myself.xviiINTRODUCTIONSince the discovery of secretin (Bayliss and Starling, 1902) at the turn of thecentury, the detection, isolation and characterization of gastrointestinal endocrinehormones has provided some unique challenges for gastrointestinal (GI)endocrinologists. Picomolar levels of circulating hormone coupled with the sparsedistribution of secretory cells has complicated the isolation of usable stocks ofbiological material. In addition, several gut hormones exist as various isoformswhich interact with a number of receptor subtypes. Therefore, following the firstcloning experiments by Jackson et a! in 1972, the evolution of molecular biologyhas provided a welcome relief to GI endocrinologists by providing a number ofnew techniques for the generation, and ultimately the manipulation and study, ofrare or novel proteins. Interestingly, little work of this nature has been done withrespect to the glucagon supeifamily of peptides.The glucagon superfamily of peptides is defined primarily by the level of sequenceconservation among its constituent members. Of late, several seemingly unrelatedpeptides have been classified within the glucagon family. These include a numberof peptides isolated from Heloderniatidae (Gila monster) venoms (Hoshino et al,1984, Parker et al, 1984, Eng Ct al, 1990, Eng et al, 1992) and pituitary adenylatecyclase-activating polypeptide (PACAP; Miyata et a!, 1989). Nonetheless,several members of the superfamily can be further characterized by their ability toact as insulin secretagogues. These include glucagon, glucagon-like peptide-1 (7-36) amide (truncated GLP- 1 or tGLP- 1), secretin, vasoactive intestinal peptide,1and gastric inhibitory polypeptide or glucose-dependent insulinotropic polypeptide(GIP). All share the ability to stimulate insulin secretion, albeit in some instancesonly in pharmacological concentrations. However, given that the combinedproperties of shared biological activity and shared sequence identity are likelyinterrelated, these may provide a rare window of opportunity to define andpossibly manipulate the insulinotropic domain of these molecules using molecularbiology techniques. Specifically, the insulinotropic domains of the two probablephysiological insulin secretagogues, GIP and tGLP- 1, probably reflect theconserved regions of the other insulinotropic members of the glucagonsuperfamily.Both GIP and tGLP- 1 consist of a relatively short amino acid sequence (42 and 29amino acids respectively) and therefore would be amenable to the generation of asynthetic gene construct for insertion into a recombinant expression system.However, tGLP-1 appears to exist in two bioactive forms: GLP-1 (7-37) andGLP- 1 (7-3 6) amide. Although there is some question regarding the relativeimportance of either of these two isoforms, the generation of the amidatedpolypeptide would be more difficult following its expression in a recombinantsystem. In addition, tGLP- 1 is relatively small and is therefore easier to generatesynthetically. GIP, on the other hand, is not post-translationally modified andtherefore can be expressed in a prokaryotic expression system. Furthermorefragment analysis of GIP (Pederson et al, 1990) suggests that the moleculecontains two separate regions: a gastric acid inhibitory region and aninsulinotropic region. Hence, the possibility existed that these two functionalitiesmight be dissected by mutational analysis. Therefore a recombinant expressionsystem for GIP was developed and the resultant clone examined by scanningmutagenesis.2GIP HISTORICAL ASPECTS-The existence of a gastrointestinally-derived mediator of insulin release has longbeen suspected. In 1906, Moore et al reported that an extract derived from porcineduodeno-jejunal mucosa improved the glycosuria of diabetic patients. Labarre(1932) coined the term “incretin” to describe the hypoglycemic agent. At thattime, only two intestinal hormones had been identified: secretin (Bayliss andStarling, 1902) and cholecystokinin (CCK) (Ivy and Oldberg, 1927). Labarre andStill (1930) demonstrated that the hypoglycemic agent was not secretin byextracting a crude preparation of intestinal mucosa several times with “acidifiedninety percent alcohol” which resulted in the generation of alcohol soluble andinsoluble fractions. The alcohol soluble fl-action was found to be a potentsecretagogue for pancreatic juice in dogs (consistent with the known biologicalactivity of secretin) but lacked hypoglycemic activity. Conversely, the insolublefraction possessed very little secretagogue activity but was found to lower bloodsugar levels. LabalTe and Still (1930) concluded that the hypoglycemic activity ofthe insoluble fl-action could not be attributed to the presence of insulin since theactivity could not be inactivated by “pepsin-hydrochloride”. Later reportsclaiming that secretin was indeed an insulinotropic factor likely resulted from theuse of pharmacological doses of the hormone since Buchanan et al (1968) wereunable to show a secretin-induced increase in insulin release (in dogs) at doseswhich produced a large increase in the flow of pancreatic juice.Conflicting reports regarding the insulinotropic activity of CCK have alsoemerged. Initial studies reporting CCK-stimulated insulin release utilized animpure preparation of the polypeptide which was later found to contain anautonomous insulinotropic factor (Brown, 1982). Subsequent studies, utilizing a99% pure preparation of CCK, reported a significant potentiation of insulin release3from the isolated perfused rat pancreas (Pederson and Brown, 1979). Nonethelessit has been suggested that CCK is not an incretin candidate since intestinal glucoseadministration is not the primary stimulus for endocrine CCK release (Creutzfeldt,1979). HOwever, this reasoning is based upon Creutzfeldt’s (1979) definition of anincretin. By Creutzfeldt’s (1979) own admission, an “incretin in the original senserefers only to endocrine transmitters for insulin release”. The glucose requirementwas later suggested by Creutzfeldt (1979) and may be an artificial parameter sincea meal solely comprised of glucose is rare.The search for a true incretin was further complicated by the work of Loew et a!(1940) who were unable to identify a hypoglycemic agent in their intestinalpreparations and concluded that prior evidence advanced to support the incretinconcept was not supported by their work. However the experiments wereperformed on fasted animals in which no attempt was made to inducehyperglycemia (Brown, 1982). This was a critical omission since it was latershown that elevated glucose levels are often required for incretin activity (Dupré etal, 1973). Nonetheless, Brown (1982) contended that the work of Loew et al(1940) had a significant negative impact on future research in this area.Therefore it was not until 1964 that interest in the incretin concept was renewed.The development of the radioimmunoassay (RIA) for insulin by Berson andYallow (1959) provided the critical tool for a re-evaluation of the incretinproblem. Based on the supposition that an endocrine link between the gut andpancreas should require the stimulation of gut endocrine cells, Elrick et a! (1964)tested the hypothesis that intestinal glucose is the stimulant of incretin release andtherefore subsequent insulin release. Hence the intravenous administration of acomparable glucose load would bypass this critical link and should not result in4comparable insulin release. Elrick et al (1964) found that oral and intravenousadministration of twenty grams of glucose to humans resulted in similar bloodglucose concentrations over the two hour experimental period. However, asignificant difference in plasma insulin levels was generated in response to the oraland intravenous glucose loads; oral glucose resulted in a significant and sustainedrise in insulin release which peaked at approximately forty minutes followingingestion and was maintained for the duration of the experiment. However,intravenous glucose administration resulted in a smaller, transient increase ininsulin release which peaked at approximately sixty minutes followingadministration and declined rapidly thereafter. Elrick and colleagues (1964)reported that this was “evidence for an additional stimulus to insulin secretion,possibly a gastrointestinal or liver factor triggered by alimentary glucose”. Theidentification of this factor began with the work of Dupré and Beck (1966). Theyfound that the administration of a partially-purified extract of CCK produced aninsulinotropic response in humans.Conflicting reports regarding the gastric acid inhibitory effect of this CCKcontaining preparation led Brown and Pederson (1970) to investigate thepossibility that an impurity might be contributing to the disparate effects. Brownet al (1969, 1970) were able to isolate an acid inhibitory factor from the partiallypurified CCK preparation and named it gastric inhibitory polypeptide (GIP)(Brown, 1971). The discovery by Dupré et a! (1973) that GIP also induced apotent insulinotropic response in humans sparked considerable interest in thehormone as a probable incretin candidate and led to the suggestion that the GIPacronym be expanded to represent the second function of the peptide; “glucosedependent insulinotropic polypeptide” (Brown and Pederson, 1976).5Unlike the CCK preparation from which it was derived, the purity of the GIPpreparation suggested that GIP was a legitimate insulinotropic factor.Nonetheless, high pressure liquid chromatography (HPLC) techniques revealedthat a truncated form of the molecule (G1P342)was present in these preparations(Brown et al, 1981). G1P342 lacks the first two N-terminal amino acids and, givenits similarity to GIP, could not be removed by conventional ion exchangechromatography or gel exclusion techniques. However, GIP3..42 was considered tobe only a minor component of the purified preparation (Brown et a!, 1981) anddoes not appear to contribute to its insulinotropic activity (Dahi et al, 1983,Schmidt et al, 1986).The physiological function of G1P342 has not been elucidated. However ahypothesis can be inferred from the work of Frohman et a! (1986, 1989) withhuman growth hormone releasing hormone (GRH). They incubated synthetichuman GRH in human plasma at a ratio of 100 jig of peptide per 1 ml of plasmaand monitored the cleavage of the molecule based on changes in HPLC profile ofthe preparation. They found that a circulating dipeptidylpeptidase (DPP) cleavedthe first two NH2-terminal amino acids from the biologically-active 44 amino acidmolecule resulting in an inactive (>1000 fold decrease) , truncated 42 amino acidmetabolite. Intravenous injection of synthetic GR}I in normal human volunteersalso resulted in the appearance (within one hour) of a plasma immunoreactiveHPLC peak coinciding with the truncated peptide. Several DPP enzymes ofdiffering specificities have been characterized. It was found that the addition ofdiprotinin A, a selective inhibitor of DPP IV, prevented the cleavage of the Nterminal tyrosine/alanine residues of GRH. Frohman et al (1989) pointed out thatboth GIP and GRH share the Tyr/Ala dipeptide at their amino terminus. Given6that, a) both are circulating polypeptides of similar size and share -a commoncleavage site for a DPP and, b) GIP3..42 is present at lower levels than GIP, G1P342(like GRH) may be an inactive metabolite of GIP and the existence of GIP3..42 maybe an artifact of normal peptide metabolism. Mentlein et al (1993), using a similarexperimental protocol, confirmed the degradation of synthetic human GIP to GIP3..42 in human plasma. Mentlein and colleagues (1993) further showed that GIP wascleaved by DPP IV in vitro suggesting that GIP3..42 may be the product ofmetabolic inactivation of GIP. Nonetheless, the lack of insulinotropic activity ofGIP3..42 does not discount the possibility of another physiological role for themolecule.DIRECT AND INDIRECT METABOLIC EFFECTS OF GIPThe physiological roles of the full-length polypeptide are better understood.Perhaps the most compelling evidence supporting the role of GIP as a majormetabolic honnone lies in its numerous direct and indirect effects on energymetabolism and homeostasis. In addition to its role as an incretin, GIP has beenshown to be glucagonotropic in the presence of low glucose levels in the rat(Pederson and Brown, 1978, Fujimoto et a!, 1978). Furthermore GIP has beenimplicated in the inhibition of lipolysis (Ebert and Creutzfeldt, 1987), stimulationof chylomicron clearance (Wasada et a!, 1981, Ebert and Creutzfeldt, 1987) andfacilitation of insulin-induced inhibition of hepatic glycogenolysis (Ebert andCreutzfeldt, 1987). GIP has also been shown to stimulate lipoprotein lipaserelease from cultured cells (Eckel et aT, 1979), possibly to facilitate triglycerideuptake into the tissues (Brown, 1991). Additionally, it has been shown that GIPimproved insulin-stimulated incorporation of palmitic acid into triglycerides in adose-dependent fashion (Beck and Max, 1983). The results implicating GIP with7fat metabolism are not surprising since long-chain triglycerides are potentstimulators of GIP release (Brown, 1974).Finally several other physiological functions have been attributed to GIP. Theseinclude a reduction in jejunal water and electrolyte absorption (Helmon andBarbezat, 1977), increase in mesenteric blood flow (Fara and Salazar, 1978) andreduction in pepsin secretion (Pederson and Brown, 1972).GIP ISOFORMSThe amino acid sequences of porcine GIP (Brown and Dryburgh, 1971; latercorrected by JOrnvall et al, 1981), bovine GIP (Cariquist et a!, 1984), human GIP(Moody et al, 1984) and rodent GIP (Higashimoto et al, 1992) are shown in Figure1. Each differs at no greater than four sites from isoforms isolated from otherspecies. This high degree of sequence conservation among species strongly arguesfor an important physiological role. This similarity is also borne out by the factthat both the human and porcine peptides exert biological actions in the isolatedperfused rat pancreas. In all species, GIP is a 42 amino acid monomericpolypeptide which does not appear to be post-translationally modified. Computersequence analysis (PC Gene: licensed from Intelligenetics, 1992) suggests onepossible amidation site at lysine(30)but there is no empirical evidence supportingthis theoretical modification.PURIFICATION OF NATIVE GIPThe site of GIP synthesis has been determined by immunohistochemical techniques.In 1975, Buffa et allocated GIP-containing cells in canine, porcine and humanintestinal mucosa using porcine GIP antisera. The GIP-containing cells hadpreviously been classified on an ultrastructural basis as “K” cells (Solcia et al,8-t, )CD— CDSC,) CDCD-jcCC)0CDCDc3.JI cr h-cJ 00‘ -(H ,c‘00,crj <4•f_ t-t->.I’ LJr.<-j tTic H r1—C/)00liD— ——— t’JJt-—1974). Like most endocrine cells, the sparse distribution of K cells-within theintact tissue complicates the isolation of the GIP molecule. Typically, theisolation of a usable stock of native GIP requires the extraction of literally severaltons of porcine gut. The purification protocol is so cumbersome, costly and timeconsuming that few laboratories are willing to cany it out routinely. However,since it remains the primary means for generating native material, it meritsdiscussion here.Briefly, the first metre of hog duodenum is collected within thirty minutes ofslaughter. The intestines are flushed with cold water, boiled then minced prior toextraction with 0.5 M acetic acid. This material is then adsorbed to alginic acid,eluted with 0.2 M HC1 and precipitated with NaCJ. The precipitate is composedprimarily of secretin, CCK and GIP. Methanol extraction results in the generationof a secretin-containing soluble fraction and GIP-, CCK-containing insolublefraction. A detailed description of the protocol to this stage has been published byMutt (1959).Jorpes and Mutt (1961) reported further purification of the methanol-insolublefraction. The fraction was first washed with ether and air-dried. Followingresuspension in water, precipitated impurities were removed by filtration and thesupernatant passed over a carboxymethyl cellulose column in 0.02 M sodiumphosphate buffer and eluted with 0.2 M NaCI. The eluate was precipitated bysaturation with NaC1. The salt cake was dissolved in 0.5% NaCl and precipitatedwith three volumes of ethanol. Two volumes of n-butanol (pre-cooled to -15°C)were added to the supernatant and the resultant precipitate washed with butanoland ether and air-dried. This was dissolved in sodiumpyrophosphateorthophosphoric acid buffer containing N,N’-dimethylformamide10and chromatographed on a triethyl-aminoethyl cellulose column and eluted withthe same buffer. The result was a “10% pure CCK” preparation containing GIP.Finally the GIP was purified from this material by Brown et al (1970). Verybriefly this entailed passage over a Sephadex G50 column, adsorption to alginicacid (elution with HCT) and passage over carboxymethyl cellulose and SephadexG25 columns. Yields are on the order of 30 mg of pure GIP (and G1P342) per100,000 hog duodena or approximately 300 jig per kilometer ofgut.Obviously the requirement for large quantities of biological starting materialprecludes the isolation of usable quantities of human GIP. Furthermore attemptsat generating a synthetic molecule have resulted in either a) a peptide of lowspecific activity or b) a peptide in which little attempt has been made to compareits biological activity to that of a naturally-derived control (Camble et al, 1973,Yajima et al, 1975, Yanaihara et al, 1978, Yajima et al 1985, Fujii et al, 1986,). Itis unclear why the synthetic human preparations did not exhibit full biologicalactivity. Early failures at the generation of a biologically-active syntheticmolecule prompted a re-examination of the reported primary structure and resultedin a minor correction to the published sequence (Jörnvall et al, 1981).Nonetheless, both synthetic and natural porcine GIP appear to be significantlymore insulinotropic than synthetic human GIP in the rat model (Xiaoyan Jia,personal con1munication).THE GIP RECEPTORAcceptance of GIP as an incretin candidate presupposes that GIP acts directly orindirectly on the pancreatic f3-cell, the site of insulin release. Perfusionexperiments on the isolated, perfused rat pancreas support this contention(Pederson and Brown,1976). Yet little is known of the nature of the pancreatic11GIP receptor nor of the critical domain(s) of the GIP molecule required-to induce areceptor-mediated insulinotropic response. Until recently, the existence of a GIPreceptor had only been confirmed in neoplastic cell lines. Specific binding of 125J..labeled GIP has been shown in a hamster insulinoma cell preparation (Amiranoffet al, 1984); Scatchard analysis was used to predict the presence of two receptorsubtypes: a high affinity, low capacity site (3,000 sites per cell) and a low affinity,high capacity site (150,000 sites per cell). Couvineau et al (1984) chemicallycross-linked 125-GIP to a crude membrane preparation which had been isolatedfrom the same cell line and identified a 125-GIP/protein complex with a molecularweight of 64,000 daltons by polyacrylamide gel electrophoresis andautoradiography. Assuming that only one GIP molecule interacts with eachreceptor, and factoring in the molecular weight of GIP, Couvineau and co-workers(1984) predicted that the molecular weight of the receptor was 59,000 daltons.The existence of a GIP receptor has also been confirmed in mouse (Verchere,1991) and human (Maletti et al, 1987) insulinomas. Maletti et al (1987) studiedbenign human insulinomas from three patients. Each tumor was divided into 2fragments; one for a perifusion procedure and the other for the generation of amembrane preparation. The perifusion, perfonned at 37°C using Krebs-Ringerbicarbonate with high (22 mM) and low (2.2 mM) glucose concentrations,confirmed that glucose potentiated insulin release at elevated levels. Specific,saturable ‘251-GIP binding to the isolated membrane preparations wascompetitively inhibited in a dose-dependent manner by GIP.Verchere (1991) was able to show‘251-GIP binding to the mouse 13TC3 cell linewhich was displaceable by porcine GIP but not by any of the structurally-relatedmembers of the secretin-glucagon family. J3TC3 cells are derived from transgenic12mice which expressed an insulin-promoted, SV4O T antigen hybrid oncogene inpancreatic 13-cells (D’Ambra et a!, 1990). Perhaps of greater significance,Verchere (1991) demonstrated 125-GIP binding to isolated rat islets. At that time,this was the only evidence for the existence of a GIP receptor in healthy pancreatictissue. However, Verchere (1991) was unable to show ‘251-GIP binding tofluorescence-activated cell-sorter (FACS)-purified pancreatic 13-cells. This waspresumably due to receptor damage associated with the additional enzymatic andcell dispersion steps required for FACS purification of 13-cells from isolated islets.Recently, a rat GIP receptor was cloned (Usdin et a!, 1993) whose message wasfound to be widely distributed in several tissues including the gut, adipose tissue,heart and brain. The authors contend that this distribution suggests that GIP mayhave previously undescribed actions. Nevertheless, the GIP receptor wasclassified in to the secretin-VIP receptor family of G-protein-coupled receptors.THE INSULINOTROPIC DOMAIN OF GIPAlthough a direct link between abnormal GIP levels and any known pathologicalcondition has yet to be shown, it would appear that GIP is involved in afundamental way in the control of serum glucose levels. In addition, the adverseeffects of aberrant serum glucose levels have been well documented. Thereforethe elucidation of the region of the molecule directly responsible for receptorligand-mediated insulin release would be useful for a number of reasons:1) The technology now exists to isolate a desired gene and determine its nucleicacid sequence. This allows for the comparison of so-called “mutant” genes withtheir wild-type counterparts and therefore allows for the identification of familialdisorders. Patients who have been previously diagnosed with an abnormal serum13glucose level and normal serum GIP level may be expressing a mutant GIPmolecule of diminished biological activity. An understanding of the amino acidsrequired for the receptor-ligand interaction would clarify whether or not geneticdisorders existed which were characterized by normal GIP levels but a GIPmolecule of reduced specific activity. If such disorders existed, a replacementtherapy could be devised using recombinant, wild-type GIP.2) The elucidation of the insulinotropic domain of GIP would be the first step inthe generation of polypeptide analogs of potential clinical significance. Forexample, agonists or antagonists of the hormone could be generated which displayenhanced or diminished circulatory half lives and therefore induce a prolonged ormuted biological response.3) GIP belongs to the glucagon family of peptides which share a fairly largedegree of sequence identity. Not coincidentally, these conserved regions probablycontribute to the insulinotropic activity of some of the family members whenadministered at pharmacological concentrations. Hence, an understanding of thebiologically-relevant amino acids of GIP may contribute to a parallelunderstanding of the biologically-active domains of the other members of thefamily.In the past, determination of the biologically-active domain(s) of GIP wasattempted in one of two ways: by proteolytic cleavage of the molecule andassessment of the biological activity of the resultant fragments or by synthesis oftruncated regions of the molecule and assessment of biological activity. Theearliest reported dissection of the insulinotropic region of the molecule was madeby Pederson and Brown in 1976. They used a cyanogen bromide digest to14generate an insulinotropic carboxy-terminal fragment colTesponding to G1P1542.Maletti et al (1986) used Staphylococcus aureus V8 protease, enterokinase andtrypsin to cleave native porcine GIP into G1P442,G1P1 6,G1P1742 and G1P1930.Biological activity was measured using a competitive binding assay between thepeptide fragments and‘251-GIP to membranes isolated from a hamster insulinomaor upon the capacity of the fragments to stimulate insulin release from the isolatedperfused rat pancreas. They reported that G1P17..42 retained both partial receptor-binding and insulinotropic capability. Given that Moroder et a! (1978) hadpreviously reported that synthetic G1P138 was fully insulinotropic, Maletti et a!(1986) concluded that the insulinotropic domain of the molecule must lie betweenresidues 17 and 38. Pederson et al (1990) reported that a synthetic G1P130fragment was also fully insulinotropic suggesting that the bioactive domain liesbetween residues 17 and 30. These results have been summarized in Figure 2.There are limitations associated with the generation and assessment ofenzymatically- or chemically-derived peptide fragments. For example, proteolyticfI-agments may retain the biologically-relevant domain(s) of the molecule but notexhibit biological activity; the removal of entire segments of the moleculepotentially disrupts its tertiary structure and hence biological integrity of thepolypeptide. Therefore, it is difficult to determine if alterations in biologicalactivity are due to the removal of interactive amino acids or of those required forstructural integrity. As such, there are definitive limits on the smallest peptidefragments which are biologically-active and therefore limits on the informationwhich can be acquired by this method. Raising a monoclonal antibody to GIPfragments and testing the ability of the antibody to immunoneutralize the intactnative peptide is one possible solution to this problem. However, given therelatively large size of an antibody, it is probable that, in some cases, an15______receptor-binding!e, Jnsulinotropicxcapacity activity______putativc bioactivedomain______________________________ 42_ __________________ _________________3_ ___ __ __ ___ __424 421617 42—1k1938315 42Figure 2: Fragment analysis of the of the GIP molecule.16interaction between the antibody and the targeted amino acids of native GIP maybe sterically impossible. Furthermore, the number and location of the cleavagesites is limited by the chance presence of target sequences and/or availability ofspecific proteases or chemical agents. Hence, the generation of defined proteolyticsequences is often difficult if not impossible, In addition, truncated fragments maybe unstable or preferentially degraded in subsequent biological assays. This wouldtranslate into an apparently low, and artificial, specific activity for the fragment.The technological ability to manipulate DNA molecules in vitro for the subsequentexpression in foreign hosts has allowed for the circumvention of these limitations.DNA manipulation techniques allow for defined and specific changes to thetranscribed and translated product be it deletion, insertion or substitution ofdesired amino acids. Furthermore, the synthesis of full-length proteins containingrelatively small and defined changes in primary sequence minimizes the possibilityof alterations in tertiary structure and maximizes the probability that recombinantGIP isoforms displaying diminished biological activity are the result of theremoval of biologically-relevant amino acids. Lastly, a full-length moleculecomprised of a predominantly native primaly sequence is likely to exhibit a similarhalf-life to the native protein and is less likely to degrade prematurely.The technology of DNA manipulation and expression in a foreign host isrelatively new. Historically, the primaiy stumbling block has been the inability tocleave and rejoin DNA molecules at defined sites. Unfortunately, the first DNAcleaving enzyme isolated from Escherichia coil by Meselson and Yuan (1968) waslater classified as a “class I” restriction enzyme which, by definition, cleaves at anill-defined site 1000 to 5000 bases downstream to its recognition site. In 1970, arestriction enzyme isolated from Haeniophilus influenzae (Kelly and Smith, 1970,Smith and Wilcox, 1970) allowed for the reproducible cleavage of DNA at a17defined nucleotide sequence. To date, hundreds of restriction enzymes -of differingspecificities have been characterized and are commercially available. Theisolation of these specific restriction endonucleases, coupled with the discovery ofa DNA ligation enzyme, facilitated the first DNA cloning experiments by Jacksonet al (1972) and Lobban and Kaiser (1973).The ability to combine disparate fragments of DNA with those containing anorigin of replication allowed for the development of the most widely-used cloningvector, pBR322 (Bolivar et al, 1977a,b) and its derivatives. The inclusion of astrong promotor system and ribosome binding site permits the preferentialexpression of a desired gene product. Obviously, given the relatively recentdevelopment of these and associated technologies, it is not surprising that the firstattempt at the generation of recombinant GIP was made only a decade ago.However, considering the cost and difficulty of obtaining GIP by classicalisolation or synthetic means, and the relative simplicity of the molecule, thedevelopment of a system for the generation of the polypeptide via recombinantmeans is attractive.RECOMBINANT GIPHorn et al (1983) first cloned and expressed a synthetic GIP gene. Chemicalsynthesis of the gene involved the selective ligation of 22 syntheticoligonucleotides, insertion of the construct into the pBR322 plasmid andexpression in Escherichia co/i strain 294. DNA sequence analysis was used toidentify a clone expressing porcine GIP. This clone was ligated to the tryptophanoperon promotor, operator, leader sequence and “E” gene. Expression of thisclone resulted in a 190 amino acid fusion protein comprised of the leader and “E”gene sequences linked to the GIP gene via a methionine residue. Leucine was18substituted for methionine at position 14 of the GIP molecule such that-it remainedresistant to subsequent cyanogen bromide (CNBr) exposure. CNBr cleavage ofthe fusion protein resulted in the release of GIPLeu 14 which was then isolated bychromatographic methods. Radioimmunoassay of the recombinant porcine GIPdemonstrated approximately 12% imniunoreactivity. Overall yields were notreported. Assays of this material in the isolated perfused rat pancreas indicatedthat it was insulinotropic. Given the subsequent advances in molecular biologytechniques, protein expression technology and recent success in the expression ofseveral proteins in bacteria, a re-evaluation of the practicality of prokaryotic GIPexpression has been undertaken.Several systems for the generation of recombinant proteins in bacteria have beendeveloped and are commercially available. These allow for the cost-effectivesynthesis of relatively large quantities of recombinant protein. Often, therecombinant protein is coupled to a short peptide sequence or a prokaryotic proteinwhich has defined binding capabilities. For example, Staphylococcus aureusprotein A, isolated from the bacterial cell wall, binds very strongly to the constantregion of immunoglobin G (IgG) from several species, including man. Hence, acovalently-coupled fusion protein can be generated which is comprised of a singleprimary sequence with two functional domains: the recombinant protein andprotein A. The fusion construct is purified from thousands of contaminatingproteins in the cell lysate by a simple passage over an IgG-Sepharose affinitycolumn and is eluted via a buffer pH change. Other commercial vectors existwhich express recombinant proteins linked to fusion moieties such as glutathioneS-transferase (GST) and j3-galactosidase (Pharmacia) which bind glutathione andp-aminophenyl- 13 -D-thiogalactoside respectively. In addition, the literatureincludes reports of several other fusion moieties such as the cellulose-binding19domain of cellulases (Ong et a!, 1989), polyarginine (purified by ion exchangechromatography; Itakura et a!, 1977), streptavidin (adsorbs to biotin; Nagai andThorgersen, 1984), protein G (adsorbs to albumin; Josephson and Bishop, 1988)and others. If desired, the fusion protein can be cleaved at a pre-engineered,proteolytic site between its recombinant and fusion domains and the nativerecombinant protein purified by chromatographic means.Although prokaryotic expression systems provide a relatively fast and cost-effective means of protein expression, there are limiting factors; prokaryotes areunable to cany out most of the post-translational modifications often required foreukaiyotic protein activity. Although GIP does not appear to be posttranslationally modified, other factors may limit the expression of high levels ofrecombinant polypeptide. Eukaiyotic protein stability and expression levels varyin prokaryotes. Determination of the best expression system is largely anempirical process and is a reflection of a limited understanding of the regulation ofprotein synthesis.SITE-DIRECTED MUTAGENESIS OF CLONED DNAIn addition to the high level expression of recombinant proteins, a DNA cloneprovides a genetic template for the creation of mutant forms of the gene andassociated development of engineered proteins. This facilitates the study ofprotein structure/function relationships. A number of techniques have beendeveloped for the introduction of mutations into cloned DNA. These can be usedto insert, delete or replace targeted amino acids in recombinant proteins withrelative ease and at a manageable cost when compared to the re-synthesis of theentire molecule.20The tTaditional approach to mutagenesis involved the generation of mutants via thetreatment of an organism with chemical or physical agents which altered the natureor composition of its DNA. This approach suffered from several disadvantages,including a) the mutation of non-target genes and b) a low mutation frequency oftarget genes, necessitating the development of selection strategies for organismsexpressing the mutation of interest. Subsequently, the technology of DNAmanipulation allowed for the in vitro mutagenesis of cloned DNA sequencesinserted into vector DNA and hence specific, defined changes to genes.Technically, the simplest mutations involve the insertion of altered, synthetic DNAsequences into naturally-occulTing restriction sites, providing that the site does notalso occur in the vector DNA. Unfortunately, the location of gratuitous restrictionsites are not always where one might desire. Unique restriction sites can beintroduced at predetermined loci by a number of methods and insertionmutagenesis performed as above. However, it is more expedient to introducemutations directly by the same techniques and circumvent the process ofrestriction site generation and insertion mutagenesis.1) Oligonucleotide-directed mutagenesisSeveral methods have been developed for the mutation of cloned DNA sequencesat defined loci. For example, in the single primer method (Gillam et al, 1980,Zoller and Smith, 1983), a synthetic oligonucleotide (containing a basemismatch{es}) primes the synthesis of the remainder of the gene (and plasmid) ona single-stranded template. The duplex product is comprised of the wild-typeDNA associated with newly-synthesized mutagenic DNA. A bacterial host strainis transformed with the heteroduplex molecule and its progeny will contain eithermutant or wild-type homoduplex DNA which can be differentiated by nucleic acidhybridization techniques. Unfortunately, most bacterial host organisms possess a21mismatch repair system which recognizes the base mismatch in the heteroduplexmolecule and theoretically will revert 50% of the mutants to a wild-typephenotype. In fact, the majority of the progeny will be wild-type since the nonmethylated, newly-synthesized strand is preferentially repaired (Kramer et al,1984).To simplify the screening process for the mutant DNA, a number of strand-selection strategies have been devised. The most popular of these methods wasdevised by Kunkel (1985). The Kunkel method strongly selects against the wild-type template strand DNA via the incorporation of a small number of uracylresidues. These templates are grown in a dut ung strain of E. co/i which lacks theability to convert deoxyuraciltriphosphate (dUTP) to deoxyuracilmonophosphate(dUMP). This enriches the cellular content of dUTP and leads to theincorporation of uracil into some of those sites normally occupied by thymine.Following the generation of a heteroduplex with this template (under conditionswhich specify normal thymine incorporation into the mutant strand), the DNA istransformed into ung+ cells. These cells remove the uracil residues from thetemplate which in turn, blocks DNA synthesis from this strand. Hence, only themutant strand is efficiently replicated.2) PCR mutagenesisA mutagenesis procedure exists which overcomes the need for specialized celllines and cumbersome strand-selection strategies. Site-directed mutagenesis ofcloned gene sequences can be accomplished using the polymerase chain reaction.In fact, a review of the recent literature revealed numerous experimentalapproaches to PCR mutagenesis of DNA (Good and Nazar, 1992, Merino et al,1992, Morrison and Desrosiers, 1993, Zhao et al, 1993). Nonetheless, virtually all22PCR mutagenesis protocols introduce a mutation(s) into the target sequence byfirst amplifying a sequence between a mutagenic primer and a primer whichanneals beyond a bordering restriction site. The second reaction extends the firstproduct beyond a restriction site associated with the opposite border. Restrictionenzyme cleavage and DNA splicing is then used to subclone the mutated cassettefor further study. The primary disadvantage of PCR mutagenesis is the infidelityof the thennostable Thernius aquaticus (Taq) DNA polymerase required for primerextension. Taq polymerase lacks 3’ to 5’ exonuclease activity and thereforemisincorporates, on average, every four hundreth nucleotide (Saiki et al, 1988)necessitating confirmation of the mutant clones by sequence analysis. However,thermostable DNA polymerases have been marketed recently which possess 3’ to5’ exonuclease activity thus reducing the likelyhood of PCR errors.ALANINE SCANNING MUTAGENESIS OF GIPIt is not necessarily clear which domains of a protein are biologically relevant andtherefore should be dissected by mutagenesis. Given the relatively large size ofmany proteins, it would be a daunting task to change all of the amino acids in theprotein of interest, purify the recombinant and test for deviations in biologicalactivity. Fortunately, a prediction of the probable bioactive domain of GIP ispossible based on two observations:1) fragment analysis of the GIP molecule suggests that the bioactive domain of GIPlies between residues 15 and 30 (Figure 2).2) GTP belongs to the glucagon family of peptides which shares a relatively largedegree of sequence identity (Table 1) and is comprised of several members which23Res.#1 23456789 101112131415GIP aiagg dir jile erserilealametGlu. his ser g dir jthr errrleuptGLP his ala g g dir j dir ser asp — ser—.jy len —Sec. bsserjg dir jthr igieugpVIP —Res.# 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30GIP ile g p pç val asn leit leu alaGlu. ser— g ala p p val g u len — asn — —tGLP —— ala ala j— — —len val — — —Sec. ser— — —— Ieu — len val — — —VIPLegend:Red shared identity with GIPGreen shared identity with a family member other than GIPRes.# residue numberGIP porcine gastric inhibitory polypeptideGlu. porcine glucagontGLP porcine human glucagon-like peptide (7-3 6) amideSec. porcine secretinVIP porcine vasoactive intestinal peptideTable 1: Amino acid sequence homologies in the glucagon family of peptides. The openboxes correspond to the positions of unique residues which are not shared among familymembers.24are insulinotropic at pharmacological concentrations (with the exception of GIPand glucagon-like peptide- 1 7-36 amide (tGLP- 1) which are insulinotropic atphysiological concentrations). Given that novel amino acid sequences within theglucagon superfamily are likely responsible for the specificity of the interactionbetween a ligand and its physiologic receptor, then the conserved sequencesamong the family members likely comprise the structural domains of the ligand.In the case of GIP, for example, this may impart structural stability in blood.However, at pharmacological concentrations, these conserved regions may beresponsible for the cross-reactivity between a ligand and a non-target receptor.Hence, inferences can be made regarding the location of the insulinotropic domainof GIP and its sequence identity with the other family members; the conservedregions of the other insulinotropic members of the glucagon family are likely to bethose which cross-react with the GIP receptor and induce an insulin response.This is consistent with the observation that the smallest bioactive GIP fragmentgenerated to date represents a region of the polypeptide with a high degree ofsequence identity with the other family members (Pederson and Brown, 1976,Moroder et al, 1978, Maletti et al, 1986, Pederson et al, 1990).Given that the probable insulinotropic domain of GIP lies within the regionflanked by residues 15 and 30, a replacement strategy can be devised whichsubstitutes the amino acids in this region with others which are not likely to mimicthe biological functions of their predecessors. Alanine substitutions are oftenfavored for this type of analysis since residue replacement with alanine eliminatesthe side chain beyond the 13-carbon and does not alter the conformation of themain chain. In addition, alanine does not impose any additional electrostatic orsteric constraints, is the most abundant amino acid, is found frequently in bothexposed and internal locations, and is found in a variety of secondary structures25(Chothia, 1976, Rose et a!, 1985). Since antigenic detenninants (Amitet al, 1987)and subunit-subunit interfaces (Argos, 1988) are dominated by side-chaininteractions, it is anticipated that the substitution of a GIP residue involved in thereceptor-ligand interaction, with alanine, will eliminate this interaction and resultin a moderation of insulinotropic activity.26SYNOPSISGlucose-dependent insulinotropic polypeptide or gastric inhibitory polypeptide(GIP) is a 42 amino acid endocrine gut hormone which exhibits several direct andindirect effects on fat and glucose metabolism. The first known, and mostscrutinized, metabolic function of the hormone was the potentiation of insulinrelease from pancreatic f3-cells in the presence of threshold glucose levels. Todate, the existence of GIP has only been confirmed in mammals but is highlyconserved, differing at no greater than four residues among species. This highdegree of sequence conservation among species suggests a major physiologicalrole.GIP is secreted from “K” cells located in the proximal intestinal mucosa. Likemost endocrine cells, the sparse distribution of “K” cells within the intact tissuecomplicates the isolation of a usable stock of the native hormone. Nonetheless,one laboratory in the world has routinely purified milligram quantities of thehormone from several tons of hog duodenum using an extensive extractionprocedure. However, the relatively recent development of DNA manipulation andexpression techniques strongly argues for the creation of a more amenable systemfor the generation of the polypeptide. GIP is not post-translationally modified andtherefore is a prime candidate for expression in prokaryotic cells. In addition, thegeneration of a GIP clone would provide the genetic template for the creation ofmutant forms of the gene and subsequent production of engineered isoforms of themolecule.27The analysis of various GIP fragments has revealed that the bioactive domainprobably lies between residues 17 and 30. This is a highly conserved regionwithin the glucagon family of peptides. Given that some of the family membersare insulinotropic in sufficient concentration, it is probable that this sharedbiological activity is the result of shared sequence identity. However furtherdissection of the bioactive domain of GIP has been limited by constraints on thesize of the peptide fragments that can maintain their biological integrity; the useof progressively smaller fragments increases the possibility that secondaryalterations to protein structure are responsible for diminished insulinotropicactivity.It is proposed that the substitution of residues in the 15 to 30 domain of the intactpolypeptide, with alanine, will minimize any deviations in tertiary structure whileremoving potentially-interactive side chains. It is hypothesized that specific aminoacid side-chains of GIP interact with the receptor and induce a receptor-mediatedintracellular response which ultimately results in the release of insulin from the 3-cell. Therefore the removal of these side-chains will result in the complete loss of,or a moderation in, insulinotropic activity. This will be accomplished by thegeneration of a prokaryotic expression system for GIP, likely via the expression ofan engineered fusion construct to simplify the purification process. Once anexpression system has been established, PCR mutagenesis of the DNA clone willbe used to generate GIP isoforms containing alanine substitutions at defined lociwithin the putative insulinotropic domain. The mutants will be purified usingconventional chromatographic techniques and will be screened in the rat pancreasperfusion bioassay.28METHODSI. PURIFICATION OF NATIVE PORCINE GIPA crude extract containing porcine GIP, supplied by Victor Mutt (Stockholm,Sweden), was purified by the method of Brown et a! (1970). Approximately 30mg of crude porcine GIP (previously designated as “EG I” (Brown et al, 1970))was applied to a 18 x 2 cm carboxymethyl cellulose-il column (Whatman, GreatBritain) and eluted with 0.01 M NH4HCO3,pH 7.05. The eluate was collected in5 ml fl-actions at a flow rate of 2.5 mi/mm. Fractions corresponding to the putativeGIP peak (EG II) were concentrated by lyophilization, and the presence of thehormone confirmed by HPLC and sodium dodecyl suiphate-polyacrylamide gel(SDS-PAGE) analysis (SDS-PAGE analysis was performed as described in sectionXV). Five micrograms of the lyophilized product was loaded on a i-BondapakC18 reverse-phase column (Waters, Milford, MA.) with a 30-45% gradient ofacetonitrile in water containing 0. 1% trifluoroacetic acid (TFA) and eluted over 10mm at a flow rate of 1 mI/mm. Detection of peptides was performed by measuringthe absorbance at 214 mm The chromatography apparatus consisted of twoWaters model 510 pumps, a Waters model 712 sample processor, a Waters model441 absorbance detector, a Waters system interface module and NEC PinprinterP6. All the components were controlled by the Maxima 820 ChromatographyWorkstation (Waters) and its associated software. Approximately 10 mg of EG IIwas applied to a 1 cm x 105 cm Sephadex G-25 fine (Pharmacia, Uppsula,Sweden) column and eluted with 0.2 M acetic acid. The eluate was collected in 129ml fractions at a flow rate of 0.7 mI/mm. Fractions corresponding to the putativeGIP peak (EG III) were analyzed as before.II. ENTEROKINASE DIGEST OF PORCINE GIP(1530)One hundred and twenty-six micrograms of synthetic porcine GIP(15..30) (a giftfrom Serge St. Pierre, IRMS Sante, Montreal, Quebec) and 550 units ofenterokinase (Sigma, St Louis, USA.) were dissolved in 250 tl of 0.02 Mphosphate buffer (0.02 M Na2HPO4,0.02 M NaH2PO4,pH 5.55), incubated atroom temperature for 1 h, then 95°C for 10 mm and concentrated bylyophilization. The lyophilized material was resuspended in 100 p1 water andloaded on a i-Bondapak C18 reverse-phase column (Waters) with a 28-38%gradient of acetonitrile in water containing 0.1% trifluoroacetic acid (TFA) andrun over 10 mm at a flow rate of I mi/mm. Fractions corresponding to each peakwere collected, concentrated by lyophilization and analyzed in the pancreasperfusion GIP bioassay.III. TRYPTIC DIGEST OF PORCINE GIP(1530)One hundred and ten micrograms of synthetic porcine GIP(15..30) and 5.5 i1 oftrypsin (Boeringer Mannheim; dissolved in 1 mM HC1 to a concentration of 1tg/jil) were combined in 94.5 p1 of 100 mM Tris-HC1, pH 8.3, and incubated at37°C for 4 hours, 95°C for 10 mm and concentrated by lyophilization. Thelyophilizate was resuspended in 100 p1 water, loaded on a ji-Bondapak C18column and analyzed as above.IV. CONSTRUCTION OF pRIT 2T HUMAN GIP (hGIP), pRIT 5 hGIPThe strategy for the construction and insertion of a synthetic hGIP gene (with anupstream Ax-fibrinopeptide sequence) into the pRIT 2T/pRIT 5 expression vectors30(Pharmacia) is summarized in Figure 3. All experiments utilizing the pRITexpression system were carried out in conjunction with Billy K.-C. Chow. PRIT 5shares the same polycloning site with pRIT ‘2T and, like pRIT 2T, expresses therecombinant gene as a protein A fusion construct. However, the pRIT 5 fusionprotein includes an NH2-terminal signal sequence which targets the recombinantfusion protein to the periplasmic space (gram negative cells) or medium (grampositive cells). Ninety-eight and one hundred base oligonucleotides synthesizedon an Applied Biosystem 380A DNA synthesizer were deprotected and elutedfrom the column with 3 ml of 14.8M NH4O , incubated at 55°C for 12 h anddried overnight on a rotary evaporator (Savant model SVC 200H centrifuge,Savant model RT 400 condensation trap and Savant model HVP83 pump).Approximately 50% of each oligonucleotide was resuspended in 30 111 of 50%(v/v) deionized formamide, heated to 68°C for 10 mm and purified byelectrophoresis on a 6% polyacrylamide gel. The slowest migrating band,identified using a hand-held, long-wavelength ultraviolet (UV) lamp, was cut fromthe remaining gel and incubated overnight in 2 ml of 0.1% sodium dodecyl sulfate(SDS) and 0.5 M ammonium acetate at 37°C. The DNA was precipitated with 2vol of isopropanol and 0.3 M sodium acetate, washed once with 80% ethanol, airdried and resuspended in 100 pi of distilled water. The DNA was then quantifiedbased on its absorbance at 260 mu in a UV spectrophotometer (Pye Unicam,Cambridge, England).The oligonucleotides contained 20 base pairs (bp) of complementary sequence attheir Y ends and were annealed as follows: one microgram aliquots of eachpurified oligonucleotide were pooled and annealed at 68°C for 10 mm inhybridization buffer (40 mM Tris-HC1, pH 7.5, 20 mM MgCl2,50 mM NaC1) thenallowed to cool slowly to room temperature. After cooling, 0.1 jiM3120 baseoverlap5’ 98 nucleotides 3’ Pst Iliii IBamIfi 3’Kienow / dNTP’sI Ac -Fibrinogen hGIP IBamHI PstIpR1T2TBarn HI I Pst I100 nucleotides 5’I’•Pstl PstlFigure 3: Construction strategy for the generation of pRIT 2T hGIP and pRIT5 hGfP.32deoxyribonucleoside triphosphates (dNTP’s), 10 mM dithiothreitol (DIT) and oneunit of DNA polymerase I Klenow fragment (Bethesda Research Laboratories(BRL), Canada) was added to the mixture in a final reaction volume of 31 ti andincubated at 37°C for 30 mm, 68°C for 10 mm and cooled slowly to roomtemperature. Five hundred femtomoles of extended product were cleaved with 5units each of Barn HI, Pst I (BRL) in React 3 buffer (BRL; 50 mM Tris-HC1, pH8.0, 10 mM MgC12, 100 mM NaCI) in a final reaction volume of 10 t1 for 60 millat 3 7°C. The restriction enzymes were removed by extraction with 0.5 vol ofphenol and 0.5 vol of chloroform, precipitated with isopropanol/sodium acetateand washed with ethanol as before. Fifty femtomoles of the pRIT 2T/pRIT 5expression vectors were cleaved at the same sites and purified in a similar manner.The DNA and amino acid sequences of the synthetic hGIP gene are shown inFigure 4.The gene construct was ligated into the cleaved expression vectors as follows: 500fmol of insert, 50 fmol of expression vector and 2 units of T4 DNA ligase (BRL)were combined in T4 DNA ligase buffer (BRL; 50 mM Tris-HCI, 10 mM MgCl21 mM adenosine triphosphate (ATP), 1 mM DTT, 5% (w/v) polyethylene glycol(PEG) 8000) in a final reaction volume of 20 tl and incubated overnight at 16°C.Ten microliters of the ligation mix was used to transform 200 jil of competent F.co/i strain DH5a (BRL) (prepared by the CaC12 method, Sambrook et al, 1989,based upon a modification of that developed by Cohen et al, 1972). Briefly, thisinvolved the addition of 10 p1 of the ligation mix to 200 p1 of ice-cold, competentE. co/i followed by a 30 mill incubation on ice and transfer to a 42°C water bathfor 2 mm. One milliliter of sterile Luria-Bertani (LB) broth (10 g bacto-tryptone,5 g bacto-yeast extract and 10 g NaC1 per liter of distilled water) was added,followed by an incubation at 37°C for 60 mi recoveiy of cells by centrifugation33fBam HI—1CGG GGA TCC TFC TTG GCT GAA GGA GGA GGA GTE AGA TAC GCT GAA GGT ACCArg Gly Ser IPhe Leu Ala Glu Gly Gly Gly Val Argi Trr Ala Glu Gly ThrHuman Fibrinogen Sequence _—Fihrombin Cleavage Sit1EEC ATC AGC GAC TAC AGC ATC GCT ATG GAC AAA ATC CAC CAG CAG GAC EECPhe fle Ser Asp Tyr Ser fle Ala Met Asp Lys fle His Gin Gin- Asp PheGTT AAC TGG TEG TTG GCT CAG AAA GGT AAA AAA AAC GAC TGG AAA CAC AACVal Asn Trp Leu Leu Ala Gin Lys Gly Lys Lys Asn Asp Trp Lys His AsnPstATC ACC CAG TAA TAA CTG CAG CTGlie Thr Gin stop stop Leu Gin LeuFigure 4: Ac-fibrinogeWhGIP construct.34(Sorval MC 12V, Delaware, USA) at 10,000 x g for 5 mill and resuspension of thecells in 50 tl of LB media. The transformed cell cultures were then spread on LBplates containing 100 pg/ml ampicillin (Sigma) and incubated overnight at 37°C.Transformants were irinoculated into 5 ml LB media, grown overnight at 37°Cwith vigorous shaking and the plasmid DNA isolated using the boiling prepmethod described by Gatermann et al (1988). The DNA sequence of the insertwas confirmed for both strands using the dideoxy chain termination method(Sanger Ct al, 1977).-V. PRIT 2T hGIP EXPRESSION OF THE PROTEIN A-hGIP FUSIONPROTEIN iN E. COLIE. coil, strain N4830-l (Pharmacia), was used as the host strain for the pRIT 2ThGIP construct. Competent N4830-1 cells were prepared as above andtransformed with the pRIT 2T hGIP construct as before. The presence of therecombinant plasmid was confirmed using the alkaline lysis method of plasmidisolation (Sambrook et al, 1989; as modified from the method of Birnboim andDoly, 1979 and Ish-Horowicz and Burke, 1981), cleavage with Barn HI/Pst I asbefore and visualization on a 2% agarose electrophoretic gel containing 0.04 MTris-acetate/0.OO1M ethylenediaminetetraacetic acid (TAE buffer) and 0.5 jig/miethidium bromide on a transillurninator (Bio/Can Scientific, Canada). Severaltransformed colonies were used to innoculate sterile LB broth containing 100jig/mi ampicillin and the culture grown to exponential phase of growth (opticaldensity at 600 nm (OD600)= 0.5-0.6) at 30°C with vigorous shaking. Thetemperature of the culture was then rapidly shifted to 42°C by adding 1 volume ofsterile LB media pre-heated to 54°C and the incubation continued for a further 5 to360 mm at 42°C.35VI. PURIFICATION OF RECOMBINANT hGIPThe cells were harvested by centrifugation at 5000 x g (Sorvall) for 10 mm at 4°Cand resuspended in 20 vol of ice-cold buffer consisting of 50 mM Tris-HC1, pH8.0, 0.1 mM DTT, 3 mM EDTA, 3 mg/mi lysozyme (Sigma), 1 unit/mIribonuclease A (Sigma), 16 units/ml of deoxyribonuclease I (Pharmacia), 0. 1 mMpepstatin A (Sigma) and 1 mM phenylmethylsuiphonylfluoride (PMSF). The cellswere then lysed by 2 passages through a french press (American InstrumentCompany, Maryland, USA) at 1200 psi. The cell debris was removed bycentrifugation at 12000 x g for 20 mm at 4°C and the contents of the pellet andsupernatant analyzed separately (summarized in Figure 5):1) Cell lysate; supernatantThe supernatant was dialyzed overnight against TST buffer (50 mM Tris-HC1, pH7.6, 150 mM NaC1 and 0.005% Tween 20) at 4°C. The dialysate was applied toan immunoglobin G (IgG)-Sepharose colunm (Pharmacia) and the column washedwith TST buffer until the absorbance readings of the eluate at 280 nm were lessthan 0.01. The unbound fraction was concentrated by lyophilization, resuspendedin TST and then re-loaded on the column to confirm that all of the fusion proteinwas removed on the first passage. The fusion protein was eluted with 0.5 M aceticacid, pH 3.4 and concentrated by lyophilization. The dried sample wasresuspended in thrombin cleavage buffer (10 mM Tris-HC1, pH 7.4, 150 mMNaCl, 10 mM Hepes, 0.0075% SDS and 1 g/l PEG 8000) and cut with x-thrombinovernight (a gift from JW Fenton II, Wadsworth Center of Laboratories andResearch, New York State Department of Health) at an enzyme to substrate molarratio of 100: 1 at 3 7°C. The cleaved fusion protein mixture was reloaded on to theIgG-Sepharose column to remove the protein A, Aa-fibrinopeptide portion of thefusion protein and other non-specific binding proteins. The unbound sample was36N4830- 1 pRIT 2T hGIP cell cultureCell Pellet Culture MediaPelletInsolubleCell DebrisE. ColiProteinsRecombinant Fusion ProteinFigure 5: Purification strategy for protein A-hGIP.6000 x g12,000xg8 M UreaSupematant12,000xgSoluble DialysatevI Urea, pH 7.6IIgG-Sepharose Column37lyophilized, resuspended in 0.2 M acetic acid and further purified bychromatography on a 1 x 20 cm Sephadex G50 fine (Pharmacia) column using 0.2M acetic acid as the buffer.2) Cell lysate, insoluble debrisTo confirm that the bulk of the fusion protein was not retained in the pellet asinclusion bodies, the following experiment was performed: the cell pellet wasresuspended in a sufficient volume of 8 M urea to ensure complete solubilization(approximately 10 vol), incubated at room temperature for 2 h with vigorousshaking and dialyzed against TST. The dialysate was spun at 10,000 x g for 30mm at 4°C and the supernatant passed over an IgG Sepharose column (Phannacia)and the bound peptide eluted with 0.5 M acetic acid, pH 3.4. The precipitate wasresuspended in 2 M urea, pH 7.6, passed over an IgG Sepharose column which hadbeen previously equilibrated with 2 M urea, pH 7.6 and eluted with 0.5 M aceticacid, pH 3.4. The yield of fusion protein was based upon absorbance readings at280 inn and upon inspection of the electrophoresis products on a 20%homogeneous polyacrylamide gel (Pharmacia) under denaturing conditions.VII. PRIT 5 hGIP EXPRESSION OF THE PROTEIN A-hGIP FUSIONPROTEIN TN F. CULlF. co/i, strain JM 101(Messing, 1979), was used as the host for the pRIT 5 hGIPconstruct. Competent JM 101 cells were transformed with the pRIT 5 hGIPplasmid as before and the presence of the recombinant plasmid verified aspreviously described. Several transformed colonies were used to innoculate sterileLB media containing 100 pg/m1 ampicillin. The culture was grown to 0D600value of 0.5-0.6 at 37°C with vigorous shaking. The cells were harvested bycentrifugation at 5000 x g for 10 mm and the periplasmic contents released by38osmotic shock by one of three separate methods: the harvested cells were, a)washed with 10 vol of 0.01 M Tris-HC1, pH 8.0, and 0.03 M NaC1, resuspended in1 vol 20% sucrose (w/v), 0.03 M Tris-HCI, pH 8.0, 1 mM EDTA, shaken at 180rpm for 10 mm and centrifuged at 12,000 x g for 10 mm at 4°C or, b) washed with10 vol 0.01 M Tris-HC1, resuspended in 1 vol 20% sucrose (w/v), 0.03 M TrisHCI, pH 8.0, 1 mM EDTA, 10 mg/mI lysozyme (Sigma) and centrifuged at 12,000x g for 10 mm at 4°C or, c) resuspended in 1 vol chloroform, incubated for 15 mmat room temperature, then 100 vol of 0.01 M Tris-HC1 added followed bycentrifugation at 5000 x g for 20 mm. In each case, the supernatant was dialyzedagainst TST and passed over an IgG Sepharose column under identical conditionsas the pRIT 2T hGIP product.VIII. CONSTRUCTION OF pGEX 2T hGIPThe strategy for the construction and insertion of a synthetic hGIPLeU 14 gene into thepGEX 2T expression vector (Pharmacia) is summarized in Figure 6. PGEX 2Texpresses the recombinant fusion gene as a glutathione S-transferase (GST) fusionconstruct. The fusion protein was engineered with an intervening methionine residuesuch that CNBr cleavage released hGIPLeU 14 A leucine residue was substituted forthe methionine residue at position 14 of the recombinant hGIP such that it remainedresistant to the CNBr exposure. Eighty-three and eighty-five base oligonucleotides(Figure 7) were synthesized on an Applied Biosystems 380A DNA synthesizer,eluted, deprotected and purified as before. In separate reactions, 30 nmol of eacholigonucleotide and I unit of RNA ligase (Pharmacia) were resuspended in T4 DNAligase buffer (Pharmacia) in a final reaction volume of 20 p1, incubated at 37°C for 30mm and transferred to a 680C water bath for 10mm. The reactions were combinedand allowed to cool to room temperature. One unit of Klenow fragment(GIBCO/BRL) and dNTPs (0.1 mM final concentration) were added in a final39Barn HII 83 nucleotides5,BarnHII 83 nucleotides5,BarnHII 83 nucleotides‘jRNA LigaseBarnHE83 83Barn HI Barn HI Barn HIEco RII!BHI0RIEcoRl85 nucleotides5,EcoRI85 nucleotides i5,EcoRl85 nucleotides iBarn HI5,jRIA Lig:eBamHI EcoRI EcoRI EcoRIi 83 85 i 85 855’I II I I IKienowdNTP’sEcoRI EcoRIFull-length clone for insertion into pGEX-2TFigure 6: Construction strategy for the generation of hGlPieu 144083 Nucleotides:5’ GGA TCC ATG TAC GCT GAA GGT ACC TTC ATC AGC GAC TAC AGCATC GCT TTA GAC AAA ATC CAC CAG CAG GAC TTC GTT AAC TG 3t85 Nucleotides:5’ GAA TTC TTA TTA CTG GGT GAT GTT GTG TTT CCA GTC GTT TTT TTTACC TTT CTG AGC CAA CAA CCA GTT AAC GAA GTC CTGCTGG3’Figure 7: Oligonucleotides used in the construction of hGIPLU 14’41reaction volume of 50 il and incubated at 37°C for 10 mm and then 68°C for 10mm. Ten units each of Barn HI and Eco RI (GIBCOIBRL) and NaC1 (100 mMfinal concentration) were added and the reaction further incubated at 37°C for 60mm. The DNA was precipitated with 2.5 111 tRNA in 2 vol of 95% ethanolcontaining 0.3 M sodium acetate. The pellet was resuspended in 20 il T4 DNAligase buffer containing 1 unit RNase A (Pharmacia), 1 unit of T4 ligase, 100 ng ofpUC 19 (GIBCO/BRL), previously digested with Barn HI and Eco RI, andincubated overnight at 16 °C. Ten microliters of this ligation mix was used totransform 200 tl of competent DH5a cells (GIBCO/BRL) as before. Forty-eightpositive clones were generated, six of which were extracted by alkaline lysis andsequenced. All six clones contained the desired full-length sequence.IX. POLYMERASE CHAIN REACTION MUTAGENESIS OF THE HUMANGIP CLONEMutagenesis of the recombinant hGIP clone was carried out by the method ofNelson and Long (1989) and is summarized in Figure 8. Briefly, a full-lengthmutagenic clone was generated as a result of 3 rounds of PCR cycling:step 1: 100 picomoles (pmol) each of mutagenic primer “A” and flanking primer“B” (Figure 9), 1 fmol of pUC 19 hGIPLeU 200 pM dNTP’s and 5 units of Taqpolymerase (BRL) were resuspended in 50 p1 of Taq buffer (BRL) and cycled 25times under the following conditions: 15 sec denaturation at 94°C, 30 secannealing step at 50°C and 1 mm elongation at 72°C. The product was isolated ona 2% agarose gel and purified by electroelution.42O—iirJ)LJ1—VIIC,)I I. aC)AxjC)V0Mutagenic Primers (“A”):hGIP Alanine 19-22:5’ GAC AAA ATC CAC GCT GCT GCT GCT GTT AAC TGG TTG 3’hGIP Alanine 23-26:5’ CAG CAG GAC TTC GCT GCT GCT GCT TTG GCT CAG AAA 3’hGIP Alanine 27-30:5’ GTT AAC TGG TTG GCT GCT GCT GCT GGT AAA AAA AAC 3’Flanking Primers:Primer “B”:5’ GGA GTA CTA GTA ACC CTG GCC CCA GTC ACG ACG TTG TAA A 3’Primer “C”:5’ CAG GAA ACA GCT ATG ACC AT 3’Primer “D”:5’ GGA GTA CTA GTA ACC CTG GC 3’Figure 9: PCR mutagenesis primers used in the generation of the alanine mutants.Mutagenic primers are 36 nucleotides in length with a 12 base central mismatch.44step 2: 600 fmol of step 1 product, 1 fmol of pUC 19 hGIPLeU 14’ 200 jtM dNTP’sand 5 units of Taq polymerase were resuspended in 50 p1 of Taq buffer and cycledonce using a 2 mm denaturation at 94°C, 30 sec annealing at 50 OC and 1 mmelongation at 72 °C.step 3: 100 pmol each of primers “C” and “D” (Figure 9) were added to the step 2reaction and 25 cycles performed under the same conditions as the step 1 reaction.Under similar conditions to those discussed previously, the product was isolatedand purified from a 2% agarose gel, digested with Barn HI and Eco RI and ligatedinto the pGEX 2T vector which had been digested with the same restrictionendonucleases. Sequence analysis confirmed the incorporation of only the desiredmutations.X. EXPRESSION OF THE MUTAGENIC hGIP/GST FUSION PROTEIN(S) INF. COLICompetent E. co/i cells were transformed as previously described and grownovernight at 37°C on LB plates containing 100 pg!ml ampicillin. Ten or morecolonies were used to innoculate a 100 ml starter culture containing 100 jig/mIampicillin which was then incubated overnight at 37°C with vigorous shaking. A25 ml aliquot of this culture was used to innoculate 1 liter of LB broth containing100 jig/mi ampicillin and the culture grown (37°C with vigorous shaking) to an0D600 reading of 0.5 to 0.6. Isopropylthio-f3-D-galactoside (IPTG) was added(0.1 mM fmal concentration) and the incubation continued an additional 4 h at37°C with vigorous shaking.45XI PURIFICATION OF THE RECOMBINANT hGIP/GST FUSION P-ROTEINSThe following steps were carried out on ice or at 4°C. The cells were harvested bycentrifugation at 5000 x g for 10 mill and the cell pellet resuspended in 10 ml ice-cold phosphate-buffered saline (PBS; 150 mM NaCI, 16 mM Na2HPO4,4 mMNaH2PO4,pH 7.3) containing 1% Trasylol v/v (Miles, Canada), 1 mM PMSF and50 mM EDTA. The cell suspension was lysed by one of 2 methods: a) a singlepassage through a french press at 1200 p.s.i.or, b) 5 successive freeze-thaw cyclesin ethanol/dry ice and a 50°C water bath. In either case the cell debris wasremoved by centrifugation at 12000 x g for 60 mm.The fusion protein was purified from the cell lysate as follows: one ml ofglutathione Sepharose sluny (Pharmacia; previously equilibrated according to themanufacturers instructions) and 1% Triton X-100 (final concentration) was addedto the lysate followed by gentle rotation at room temperature for 30 mm. Thematrix was isolated by centrifugation at 500 x g for 5 mm and washed 3 x with 10bed volumes of PBS. The fusion protein was eluted with 1 bed volume of 20 mMreduced glutathione (Pharmacia) in 50 mM Tris-HC1, pH 8.0, and the matrixremoved by centrifugation at 500 x g. The supernatant was set aside on ice andthe elution step repeated a further 3 times. The 4 eluates were pooled, dialyzedagainst distilled water at 4°C, and concentrated by lyophilization.XII. OPTIMIZATION OF GST/hGIP FUSION PROTEIN EXPRESSIONGST/hGIP expression levels were tested in seven strains of E. coil, DH5a andTopp 1, 2, 3, 4, 5 and 6 (Stratagene, La Jolla, California), at various inductiontimes:461) Effect of bacterial strain on protein expression levels:-PGEX 2T hGIPLeu was used to transform the competent E. co/i strains asdescribed earlier. Each was grown overnight at 37°C on LB plates containing 100jtg/ml ampicillin. Ten or more colonies from each strain were used to iimoculate a4 ml starter culture of LB containing 100 pg/ml ampicillin which was grownovernight at 37°C with vigorous shaking. Seven hundred and fifty microliters ofstarter culture was used to innoculate 14.25 ml of LB broth + ampicillin and theculture grown as before to an 0D600 value of approximately 0.4 (Table 2). IPTGwas added to each (0.1 mM final concentration) and the incubation continued anadditional 2 h at 37°C with vigorous shaking. The cells were recovered bycentrifugation at 5000 x g for 10 mm, resuspended in PBS buffer, pH 7.3,containing 1% Trasylol, 1 mM PMSF and 50 mM ETDA and lysed by 5successive freeze/thaw cycles in ethanol/dry ice and 50°C water baths. Cell debriswas removed by centrifugation at 12000 x g for 20 mm at room temperature andthe supernatant dried in a Speed-vac rotary evaporator. The samples werequantified by SDS-PAGE and western blot analysis.Bacterial Strain Culture TimeDH5cc 5 h, 10 mm 0.356Topp 1 5 h, 10 mm 0.393Topp2 4h 0.399Topp 3 3 h, 25 mm 0.403Topp 4 3 h, 25 mm 0.420Topp5 4h 0.393Topp6 5h, 10mm 0.401Table 2: E. co/i culture densities at harvest.472) Effect of induction time on protein expression levels:-A 100 ml Topp 3 pGEX 2T hGIPLeU , culture was grown, as above, to an 0D600value of 0.543. IPTG was added (0.1 mM final concentration) and the culturesubdivided into seven 10 ml aliquots and incubated for various induction timesbetween 30 mm and 24 h. The cultures were harvested, lysed and analyzed asbefore.XIII. CYANOGEN BROMIDE CLEAVAGE OF THE GST/hGIP FUSIONPROTEINSThe GST/hGIP fusion proteins were cleaved at molar ratios ranging from 100:1 to14,000:1 of CNBr to recombinant protein in 70% formic acid at room temperaturefor 6 to 28 h. The cleaved protein was recovered in a Speed-Vac rotaly evaporatorat room temperature.XIV. PURIFICATION OF hGIPLeu 14 hGIP 14 ALAN1NESUBSTITUTED RECOMBINANT PROTEINS1) Purification Of hGIPLeU 4.Following the CNBr cleavage and lyophilization of the GSThGIPLefl 14 fusionconstruct, the cleavage product was purified as follows: approximately 5 mg oflyophilized protein was resuspended in 200 tl of distilled water and centrifuged at12,000 x g for 5 mm at room temperature. The supernatant was loaded on a p.Bondapak C18 reverse-phase colunm (Waters) with a 28-35% gradient ofacetonitrile in water containing 0.1% TFA and run over 10 mm at a flow rate of 1mi/mm. Fractions were collected across the elution profile, dried by lyophulizationand examined .by SDS-PAGE and western blot analysis. Fractions containingimmunoreactive material were resuspended in 50 mM phosphoric acid + 10 mM48NaC1, pH 6.0, at a final protein concentration of 1 mg/mi, and loaded on asuiphapropyl column (Waters; Protein Pak SP 5PW). All of the hardware andassociated software used for the generation of the gradient was the same as thatused for the reverse phase purification and analysis of the recombinants. Theprotein was eluted with a 10-75 mM NaC1 gradient in 50 mM phosphoric acid, pH6.0, at a flow rate of 1 ml/min. Fractions corresponding to the peaks werecollected, concentrated by lyophilization, resuspended to saturation in distilledwater and desalted by a second passage over a C18 reverse phase column with a 0-50 % gradient of acetonitrile in water containing 0.1% TFA and run over 10 mm ata flow rate of 1 mi/mm. Fractions corresponding to the protein peak(s) were driedby lyophilization and examined by SDS-PAGE and western blot analysis.2a) Purification of hGIP 14, Ala 23-26’ protocol 1:Given that hGIPLeU 14, Ala 23-26 has the same isoelectric point as hGIPLeU 14’ thepurification protocol for this recombinant was virtually identical to that ofhGIPLCU 14’ However, in an attempt to maximize yields, an alternative purificationprotocol was developed.2b) Purification of hGIPLeu 14, Ala 23-26’ protocol 2:2b. 1) Thrombin cleavage of GSThGIPLeu 14, Ala 23-26One mg of GST-hGIPLCU 14. Ala 23-26 was resuspended in 100 jil of thrombinreaction buffer (10 mM tris-HCI, pH 7.4, 150 mM NaCI, 10 mM Hepes, 0.0075%SDS, 1 g/l PEG 8000). Six units of bovine thrombin (Sigma) were resuspended in10 ml of thrombin reaction buffer and the reaction mixtures combined andincubated at 37°C for 3 h.492b.2) Purification of the GSThGIPLeu 14, Ala 23-26 digestion products:One volume of 8 M urea was added to the digestion mixture, which was thenvortexed and centrifuged at 12000 x g for 5 mm at room temperature. Thesupernatant was passed over a Sep-pac C18 cartridge (Waters) which had beenpreviously equilibrated with a) 5 ml acetonitrile containing 0.1% TFA and b) 5 mlof 4 M urea. The cartridge was washed with 10 ml of distilled water containing0.1% TFA and then 5 ml of 20% acetonitrile containing 0.1% TFA. The proteinwas eluted with 3 ml of 40% acetonitrile containing 0.1% TFA and the solventremoved by lyophilization. The theoretical product of the thrombin digest wascomposed of a short amino terminus glycine-serine-methionine (gsm) peptidelinked to hGIPLeu 14. Ala 23-26 and therefore will be referred to as “gsm-hGIP 14,‘IAla 23-262b.3) CNBr cleavage and HPLC purification of gsm-hGIP 14, Ala 23-26Approximately 400 pg of gsm-hGIP 14, Ala 23-26 was cleaved at a 50: 1 molarratio of CNBr to recombinant protein in 300 p1 of 70% formic acid at roomtemperature for 6 h. The cleaved protein was recovered by rotary evaporation atroom temperature and loaded on a p-Bondapak C18 reverse-phase column(Waters) with a 28-35% gradient of acetonitrile in water containing 0. 1% TFA andrun over 10 mm at a flow rate of 1 mI/mm. Fractions were collected across theelution profile, dried by lyophilization and examined by SDS-PAGE and westernblot analysis.3a) Purification Of hGIPLeU 14. Ala 19-22 and hGIPLeu 14, Ala 27-30’ protocol 1:Following the CNBr cleavage and lyophilization of the GSThGIPLeu 14. Ala 19-22and hGIPLU 14. Ala 27-30 fusion constructs, the cleavage products were partiallypurified as follows: approximately 5 mg of lyophilized protein was resuspended in50200 l of distilled water and centrifuged at 12,000 x g for 5 mm at roomtemperature. The supernatant was loaded on a i-Bondapak C 18 reverse-phasecolumn (Waters) with a 28-35% gradient of acetonitrile in water containing 0.1%TFA and run over 10 mm at a flow rate of 1 ml/min. Fractions were collectedacross the elution profiles, dried by lyophilization and examined by SDS-PAGEand western blot analysis.3b) Purification Of hGIPLeU 14, Ala 19-22 and hGIPLeU 14. Ala 27-30’ protocol 2:Alternatively, both GSThGIPLeu 14. Ala 19-22 and GST-hGIPLCU 14, Ala 27-30 werecleaved with thrombin and purified under similar conditions to the GSThGIPLeu14, Ala 23-26 recombinant protein (refer to section 2b. 1 to 2b.3). However, theenzyme to substrate ratio was increased by as much as 100-fold and the digestsallowed to proceed for 3 to 18 h. The cleavage products of the CNBr-treated gsmproteins were loaded on a i-Bondapak C 18 reverse-phase column (Waters) with a33-40% gradient of acetonitrile in water containing 0.1% TFA and run over 10mm at a flow rate of 1 mllmin.XV. SODIUM DODECYL SULPHATE-POLYACRYLAMIDE GELELECTROPHORESISProtein samples were resuspended in 2.5% beta-mercaptoethanol, 5% SDS andboiled for 2 mm. SDS gel electrophoresis, Coomassie and silver stains wereperformed in the Pharmacia PhastSystem using 8-25% gradient Phastgels (fusionprotein resolution) and high density Phastgels (recombinant hGIP resolution)purchased from Pharmacia and used according to the manufacturers instructions.51XVI. ENZYME-LINKED IMMUNOSORBENT ASSAYS (ELISA)The protocol for ELISA analysis was based upon by the method of Voller et al(1976).1) Sensitivity of ct-GIP(7-5-7) to hGIP and porcine GIP:step I) Lyophilized samples of recombinant and native porcine GIP werereconstituted in carbonate-coating buffer (1.59 gIl Na2CO3,2.93 gIl NaHCO3,0.2gil NaN3) at a final concentration of 1 igIml. One hundred microliter aliquots ofthe samples were then added (in duplicate) to a 96 well plate (Falcon 3912) andincubated overnight at 4°C.step 2) The coating solution was removed by inversion and the residual solutionremoved with 3 washes of PBS-Tween. One hundred microliters of blockingagent (5% fetal calf serum in PBS-Tween) were added to each well and the plateincubated at room temperature for 90 mm. The blocking solution was removed byinversion of the plate and each well washed 3X with PBS-Tween. One hundredmicroliter aliquots of various dilutions of a-GIP(7-5-7) ascites (containing an antiGIP monoclonal antibody; raised in mice and prepared by others at thislaboratory) was added followed by a further incubation for 1 h, removal of thebuffer and wash as before.step 3) A one hundred microliter aliquot of a 1:1000 dilution (in PBS-Tween) ofgoat anti-mouse IgG (Tago, Burlington, CA) was added to each well. The platewas incubated in the dark for 1 h and the unbound conjugate removed by inversionof the plate followed by 3 washes with PBS-Tween.52step 4) One hundred microliters of phosphatase substrate was added to each welland the reaction allowed to proceed in darkness at room temperature. Thephosphatase substrate was prepared by adding a 5 mg tablet of Sigma 104-Phosphatase Substrate to 5 ml of diethylolamine buffer (97 ml diethylolamine, 0.2g NaN3, 100 mg MgCl2-6H0, pH 9.8). The color development was monitoredand quantified at 405 urn on a Microelisa Autoreader MR580 (DynatechLaboratories, Alexander, VA).2) Confirmation of hGIP immunoreactivity by ELISA:Using the same protocol described above, the imrnunoreactivity of the recombinanthGIP was confirmed. However, fixed primary antibody concentrations (1:2000dilution of ascites) were used against various known concentrations ofrecombinant hGIP. Another monoclonal antibody, soma-lO (prepared in thislaboratory), was used as a control to test for non-specific cross-reactivity andremnant protein A activity in the purified sample of recombinant protein.XVII. WESTERN BLOT ANALYSISPhastgel system (Pharmacia) polyaciylamide gel electrophoresis of proteinsamples and subsequent transfer to a nitrocellulose membranes (Schleicher andSchuell, Keene, New Hampshire) were performed according to the manufacturer’sinstructions. The proteinlnitrocellulose complex was then blocked by immersionin 1% (w/v) gelatin in TBST buffer (10 mM Tris-HCI, 150 mM NaC1, 0.05%Tween-20, pH 8.0) for 30 mm at room temperature with gentle agitation. Apolyclonal rabbit anti-porcine GIP antibody, LMR 34 (a gift from Lynda Morgan,University of Surrey, Guilford, U.K.), was added at a final plasma:buffer dilutionof 1 in 20,000 and the incubation continued a further 60 mm at room temperaturewith gentle agitation. The membrane was washed with TBST buffer, immersed in53TBST/gelatin containing a 1:5000 dilution of goat anti-rabbit IgG/alkalinephosphatase conjugate (Jackson Immuno Research, West Grove, Pennsylvania)and incubated for 60 mm as before. Following a final wash in TBST, colordevelopment was monitored in 5 ml alkaline phosphatase buffer (100 mM TrisHC1, 100 mM NaCI, 5 mM MgCI2, pH 9.5) containing 33 tl of nitrobluetetrazolium chloride (NBT) and 16.5 tl of 5-bromo-4-chloro-3-indolylphosphatep-toluidine salt (BCIP) substrates (Immuno Select; GIBCO/BRL). The reactionwas terminated by immersion of the membrane in 20 mM Tris-HC1, 5 mM EDTA,pH 8.0.XIII. AMINO ACID SEQUENCE ANALYSIS OF RECOMBINANT PROTEINSThe purified recombinant hGIP mutants were resolved on a 15% SDSpolyacrylamide gel and were electroblotted on to an immobilon membrane(Matsudaira, 1987) and subjected to gas-phase amino acid sequence analysis. Theanalysis was performed on the Applied Biosystems 477A Protein Sequencer by theProtein Microchemistry Center at the University of Victoria, British Columbia,Canada.XIX. PANCREAS PERFUSION GIP BIOASSAYThe pancreas perfusion assay and subsequent insulin radioimmunoassays (MA)were performed by Dr. Raymond Pederson as follows: overnight fasted Wistarrats were anesthetized with pentobarbital (60 mg/kg) and the pancreassubsequently isolated as described previously (Pederson and Brown, 1976). Theperfusate consisted of modified Krebs-Ringer bicarbonate buffer containing 3%dextran and 0.2% bovine serum albumin (RIA grade) gassed with 95%°2’CO2 to achieve a pH of 7.4. Following a 10 mm equilibration period, portal veineffluent was collected at 1 mm intervals at a rate of 4 ml/min. At time 0, perfusate54containing 16.7 mM glucose was introduced along with a GIP gradient of 0-1nglml. This was accomplished by the use of a gradient apparatus consisting of twoconnected, identical perfusion flasks of equal cross section, the flask distal to thepancreas containing GIP and the proximal flask containing GIP-free perfusate.This apparatus had previously been shown to deliver a linear gradient ofsecretagogue added to the distal flask (Pederson et al, 1982). Immunoreactiveinsulin was measured by radioimmunoassay employing iodinated bovine insulinand rat insulin as standard (Pederson and Brown, 1976). The antiserum used wasa lyophilized preparation of anti-human insulin serum (guinea pig).XX. EXPRESSION OF RESULTSInsulin release data were calculated as mean and standard error of the mean.Significance was determined using the Student’s t test and statistical significancewas set at the 5% level. All integrated insulin responses were calculated from thebeginning of the glucose infusion (time=6 mm of the peifusion experiments).55RESULTSI. GIP FRAGMENT GENERATION:Ia) Enterokinase digest of synthetic porcine G1P1530A synthetic porcine GIP fragment, colTesponding to residues 15 to 30 of the native42 amino acid molecule, was digested with enterokinase for periods ranging from2 mm to 15 h and the cleavage products resolved on a C18 reverse phase columnover a 28-38% gradient. Theoretically, enterokinase should remove the aminoterminal aspartic acid and lysine residues from the synthetic fragment (Figure 10).Following the 60 mm digestion, at least 9 distinct peaks were resolved (Figure1 1C); two peaks with retention times of 8.41 mm and 13.19 mm co-eluted withenterokinase (which had been incubated under identical conditions) andundigested G1P1530 respectively (Figure 1 1A, B and C). The remaining 7 peakswere apparent products of the enterokinase digestion.Tb) Tivptic digest of synthetic porcine GIP1530 purification and sequenceanalysis of the product:A synthetic porcine GIP fragment, conesponding to residues 15 to 30 of the native 42amino acid molecule, was digested with trypsin for 4 h at 37°C and the cleavageproducts resolved on a C18 reverse phase column over a 28-38% gradient.Theoretically, the tiypsin digest should remove the amino terminal aspartic acid,lysine, isoleucine and arginine residues from the synthetic fragment (Figure 10). Theresultant profile was comprised of at least 2 novel and distinct5615 30Asp Lys lie Arg Gin Gin Asp Phe Val Asn Trp Leu Leu Ala Gin LysI tTpsjnEnterokinaseFigure 10: Cleavage sites in the 15 to 30 domain ofporcine GLP.5700Figure 11: Reverse phase HPLC profiles of porcine G1P1530 prior to (B), andfollowing (C), the enterokinase digest. Each was resolved on a 28-38% gradient ofacetonitrile in water containing 0.1% TFA which ran from 9 to 19 mm on thechromatograms. The elution profile of enterokinase is shown in A.0.4BMiNUTES X0.10MiNUESX0.4IsIINUTES X 1O258peaks (Figure 12C) which did not co-elute with trypsin (Figure 12A) norundigested G1P1530 (Figure 12B). The peak which eluted at 14.55 mm wassubjected to gas phase sequence analysis by the Protein Microchemistry Center atthe University of Victoria, British Columbia, Canada. The sequence correspondedto residues 19 to 29 of the GIP molecule. A designation for the residuecorresponding to GIP residue 30 was not possible.II. ANALYSIS OF THE GIP FRAGMENTS:ha) Pancreas perfusion bioassay of the enterokinase digestion productsThe individual peaks which comprised the C18 elution profile (Figure 11) of theenterokinase digest were collected and concentrated by lyophilization. Each wasperfused as a gradient of 0-1 ng/ml and analysed for its ability to potentiate insulinrelease in the isolated perfused rat pancreas. The results were compared toperfusions of undigested G1P1530 and native porcine GIP under identicalconditions. Only peak 3 was able to significantly potentiate insulin release abovethat of the glucose control (Figure 13). However, given the lower molecularweight of the GIP fragments when compared to the full-length molecule, these andall subsequent fragment perfusions were performed with an approximately 4-foldhigher concentration of peptide when considered on a molar basis.lib) Pancreas perfusion bioassay of synthetic G1P1730A synthetic porcine GIP fragment, colTesponding to residues 17 to 30 of the native 42amino acid molecule, was perfused through the isolated rat pancreas as a gradientfrom 0 to 1 ng/ml with a perfusate concentration of 16.7 mM glucose. The resultswere compared to perfusions of undigested G1P15..30 and native porcine GIP underidentical conditions (Figure 14). The synthetic G1P1730 fragment significantly59-J0>-J0>MZINUrES X 10Figure 12: Reverse phase HPLC profiles of porcine G1P1530 prior to (B), andfollowing (C), the tiypsin digest. Each was resolved on a 28-38% gradient ofacetonitrile in water containing 0.1% TFA which ran from 9 to 19 mm on thechromatograms. The elution profile of trypsin is shown in A.uN1xrEs X 1OMINurES x iO’03c606000 *Figure 13: Effect of a linear gradient of enterokinase-derived peak 3 on the insulinresponse of the isolated perfused rat pancreas. In this and all subsequent perfusionexperiments, the peptides were infused as a linear gradient from 0 to ing ofpeptide/mi of perfusate; all of the surgical preparations and assays were performed byDr. Raymond Pederson and Leslie Checknita.Inset: Integrated insulin responses of the glucose control, peak 3, G1P15..30 andporcine GIP compiled from the same data. All insulin responses were compared tothe release associated with glucose alone.* p <0.05, G1P15..30 n=2, porcine GIP n=3, glucose n3 peak 3 n3.5000El Glucose1111 GIP 15-30Peak3Porcine GIP4000300020001000—D——— Glucose0 Porcine GIP----•--• Peak3----A---- GIP 15-300 10 20 30Time (mm)4061Figure 14: Effect of a linear gradient of synthetic GTP17..30 on the insulin responseof the isolated perfused rat pancreas. Inset: Integrated insulin responses of theglucose control, G1P1730 G1P1530 and porcine GIP compiled from the same data.All insulin responses were compared to the release associated with glucose alone.* p <0.05, G1P1730 n=4, porcine GIP n=3, glucose n=3 G1P1530 n=2.4030-20-EzGlucoserTTr10-GIP 15-30GIP 17-30Porcine GIP400030002000:iz—D-——— Glucose0 Porcine GIP- --- •---- GIP 17-30----A---- GIP 15-3000 10 20 30 40Time (mm)5062potentiated insulin release above that of the glucose controls over the perfusionperiod from 14 to 41 mill. Although G1P15..30 was found to lack insulinotropicactivity, the result of the G1P1730 perfusion confirmed that the removal of amino-terminal aspartic acid and lysine residues restored partial insulinotropic activity ofthe molecule when compared to the full-length native 42 amino acid molecule. Inaddition, the similar biological activities of the synthetic GIP1730 and peak 3 ofthe enterokinase digest suggested that peak 3 may have had a similar primarysequence to the 17 to 30 domain of GIP (Figure 15).-TIc) Pancreas perfusion bioassay of G1P19..30The product of the trypsin digest of GIP15.30,G1P1930,was perfused through theisolated rat pancreas as a gradient from 0 to 1 ng/ml with a perfusate concentrationof 16.7 mM glucose. The results were compared to perfusions of a glucose controland native porcine GIP (Figure 16). The G1P1930 fragment significantlypotentiated insulin release above that of the glucose controls (Figure 16).III. PROKARYOTIC EXPRESSION OF HUMAN GIP AS A PROTEIN-AFUSION PROTEINlIla) Expression of human GIP as a Saphylococcus aureus protein A fusionprotein in Escherichia coilA synthetic human GIP gene, coupled to a 5-prime human Act-fibrinopeptidesequence, was inserted into the commercial pRIT 2T vector which expresses therecombinant insert as a protein A fusion construct. Expression of the fusion proteinwas controlled by a R temperature-sensitive repressor and its associated promotor.A temperature shift, from 37 to 42°C, of the transformed E. coil strain N4830-1 cellculture resulted in the expression of a 35 kDa product as indicated in the SDSpolyacrylamide gel (Figure l7A lanes 5, 6 and 7). The protein band was not63—0--- Glucose200040Figure 15: A comparison of the insulin responses of the isolated perfused ratpancreas to enterokinase peak 3 and synthetic porcine G1P17..30 Glucose n=6,porcine GIP n=3, peak 3 n=3, G1P15.30 n=2, G1P1730 n=4.640 Porcine GIPPeak 3----k--- GIP 15-303000GIP 17-301000•00 10 20 30Time (mm)C’)50Figure 16: Effect of a linear gradient of G1P19.30 on the insulin response of theisolated perfused rat pancreas. Inset: Integrated insulin responses of the glucosecontrol, G1P19..30 and porcine GIP compiled from the same data. All insulinresponses were compared to the release associated with glucose alone.* p <0.05, G1P19.30 n=9, porcine GIP n=8, glucose n12.E GlucoseJIll GIP 19-304000—El-——— Glucose+ GIP 19-30- -- 0---- Porcine GIP100000 10 20 30 40Time (mm)65A B94.6743—3O2O_14 _14_8.26.2Figure 17: SDS-PAGE analysis (12% homogeneous) of the expression andpurification of recombinant hGIP from the pRIT 21 expression system.(A) Lanes,1, 8: molecular weight markers2: total protein from N4830-1 cells cultured at 30°C3: total protein from N4830-l cells cultured at 42°C4: total protein from N4830-l/pRIT 2T hGIP cultured at 30°C5: total protein from N4830- 1/pRIT 2T hGIP cultured at 37°C6: total protein from N4830-l/pRJT 2T hGIP cultured at 42°C7: purified fusion protein following the first passage over the IgGSepharose column; the closed triangle indicates the putative fusionprotein(B) Lanes,1, 7: molecular weight markers2: fusion protein following 1 h thrombin exposure3: fusion protein following 3 h thrombin exposure; open triangle indicatesthe released hGIP4: cleaved fusion protein following a second passage over the IgGSepharose column5: purified recombinant GIP following passage over a Sephadex G50column6: natural porcine GIP1 2 3 4 5 6 71 2 3 4 5 6 7 8: I .,_ —67.343.1 !—066seen in the non-induced culture nor in the culture lacking the expression vector(Figure 1 7A, lanes 2, 3 and 4). Analysis of the insoluble debris resulting from thecell lysis indicated that 90 % of the fusion protein was contained in the cell lysatesupernatant. The fusion protein was purified on a one-step immunoaffinitycolumn by taking advantage of the strong binding between the constant region ofhuman IgG and the protein A domain of the fusion protein. However, passageover the Sepharose-coupled human IgG column resulted in the co-purification of anumber of non-specific contaminant proteins (Figure 17A, lane 7).A parallel expression was performed in the pRIT 5 expression system. Thissystem is virtually identical to the pRIT 2T system except that the translatedprotein A fusion construct contains an amino-terminus signal sequence whichtargets the recombinant to the media (gram positive cells) or periplasm (gramnegative cells). However, expression levels of the recombinant in this systemwere negligible and, given the superior expression levels in the pRIT 2T system,the use of the pRIT 5 vector was not pursued further.Ilib’) Enzymatic cleavage of the protein A-hGTP fusion protein with thrombinFollowing the partial isolation of the fusion protein by immunoaffinitychromatography, cc-thrombin cleavage of the Ax-fibrinopeptide domain was used torelease the GIP moiety. As indicated by the disappearance of the 35 kDa fusionprotein band and associated appearance of 31 and 4 kDa bands, the reaction wascompleted in 3 h at 37°C (Figure 17B, lanes 2 and 3). However, 0.0075% SDS in thereaction buffer was required to unfold the fusion protein, presumably to allowenzymatic access for protein cleavage (Figure 18). Varying ionic strength from 0 to125 mM NaC1 did not effect cleavage efficiency. The cleaved fusion protein mixturewas re-chromatographed once more through the IgG-Sepharose column to remove67941 2 3 4 5 6 7 86743302014Figure 18: SDS-PAGE analysis (20% homogenous) of the SDS requirements for thethrombin digestion of protein A-hGIPLane,1: molecular weight marker2: thrombin + 0.0075% SDS3: thrombin + 0.0 1% SDS4: uncut protein A-hGIP5: protein A-hGIP + thrombin6: protein A-hGIP + thrombin + 0.0075% SDS; arrow indicates thereleased hGIP7: protein A-hGIP + thrombin + 0.0 1% SDS8: natural porcine GIP—V68the protein A-fibrinopeptide product and other non-specific binding proteins whichco-eluted with the fusion protein in the first column. At this stage, therecombinant hGIP represented approximately 10% of the total protein in thesample (Figure 17B, lane 4). Further chromatography on Sephadex G50 fineresulted in the purification of the recombinant product to near homogeneity bySDS-PAGE (Figure 17B, lane 5) and HPLC profiles (Figure 19A, B). Theretention times of the natural porcine GIP and recombinant hGIP were 15.33 and15.27 minutes respectively. In addition, the native porcine GIP profile contains adocumented G1P342 peak (Brown et al, 1981) with a retention of 15 mm. Theabsence of this peak in the recombinant hGIP HPLC profile suggests that thispreparation lacks this contaminant. The yield of fusion protein and recombinanthGIP from a one liter culture was approximately 5 mg and 100 jig respectively.Ilic’) ELISA analysis of recombinant hGIPThe immunoreactivity of the recombinant hGIP was confirmed using cc-GIP(7-5-7), a mouse anti-porcine GIP monoclonal antibody. The ELISA results indicatedthat the affinity of cL-GIP(7-5-7) to the recombinant product was specific andcomparable to porcine GIP (Figure 20A). Another monoclonal antibody, soma-lO,a mouse anti-somatostatin monoclonal, was used to test for non-specific crossreactivity and remnant protein A in the purified sample (Figure 20B).hid) Amino acid sequence analysis of recombinant GIPThe cleavage specificity of thrombin to the fusion protein was confirmed by NH2-terminus amino acid sequence analysis of the cleaved product. The first 29 aminoacids of the purified recombinant were identical to wild-type hGIP with the exceptionof residue 18 where leucine replaced histidine (Table 3). DNA sequence analysis69cnriRetention Time (mm.)25.0Figure 19: Reverse phase HPLC profiles of porcine (A) and recombinant humanGIP (B) resolved on a 0-50% gradient of acetonitrile in water containing 0.1%TFA. The gradient ran from 9 to 19 miii on the chromatogram followed by a washin 70% acetonitrile.A -02A2 5B(‘4tr5.0 10.0 15.0 20.0I I70A0 rhGP10C GP (7.5-7) concentratIon (ItutIon)B2—— CIP7-5-7e0•1hGIP(ngfml)Figure 20: Jnimunoreactivity of recombinant hGIP. (A) Sensitivity of oGfP(7-5-7) to hGIP and porcine G[P. ELISA plates were coated with 1 igJ..d of antigenand then incubated with various dilutions of monoclonal antibody. (B) ELISAplates were coated with known concentrations of recombinant hGFP and incubatedwith a 2000-fold dilution of the antibody (ascites).71Cycle Residue Yield— Cycle Residue Yield(pmol) — (pmol)1 Tyr 7.04— 16 Lys 1.452 Ala 13.59— 17 lie 2.433 Glu 6.99 18* Leu 2.054 Gly 6.10— 19 GIn 2.115 Thr 6.12— 20 Gin 3.196 Phe 8.50— 21 Asp 0.997 lie 5.46— 22 Phe 1.488 Ser 2.55— 23 Val- 1.119 Asp 3.14— 24 Asn 1.4310 Tyr 3.57— 25 Trp 0.4611 Ser 2.16— 26 Leu 1.6812 lie 2.64— 27 Leu 2.3013 Ala 3.20— 28 Ala 0.9814 Met 2.05 — 29** Gin 1.5415 Asp 1.93—acid 18 is histidine instead of leucine.**After cycle 29 assignments are tentative.Table 3: Result of gas phase sequence analysis of recombinant hGIP.72of the clone confirmed that this substitution was an artifact of amino acidsequencing.Ille) Bioassay of recombinant hGIPBoth the recombinant hGIP and native porcine GIP were perfused through theisolated rat pancreas as a gradient from 0 to 1 ng/ml with a perfusate concentrationof 16.7 mM glucose. When the variability of the insulin responses from individualexperiments was taken into consideration, there was no significant differencebetween the insulin responses to the two peptides (Figure 21).IV. PROKARYOTIC EXPRESSION OF HUMAN GIP AS A GLUTATHIONES-TRANSFERASE FUSION PROTEFNIVa) Expression of human GIP as a Schistosorna Japonicuni glutathione Stransferase fusion protein in Escherichia co/iA synthetic human GIP gene, with a 5-prime methionine codon and methionine toleucine substitution at the codon encoding GIP residue number 14, was inserted intothe commercial pGEX 2T vector. Gene insertion and subsequent protein expressionfrom pGEX 2T results in the generation of a glutathione S-transferase (GST) fusionconstruct. Expression of the fusion protein was driven by the IPTG-inducible Ptacpromotor. The addition of IPTG to a culture which had been previously transformedwith pGEX 2ThGIPLeu 14 resulted in the expression of a novel 29 kDa fusion proteinproduct which was not seen in a culture which had been transformed with pGEX 2Talone (Figure 22A!B, lane 4). The fusion protein was purified using affinitychromatography by taking advantage of the strong interaction between the glutathioneS-transferase moiety of the fusion protein and glutathione-Sepharose. The yield735000zPorcine GIP50Figure 21: Effect of a linear gradient of recombinant human GIP or porcine GIP onthe insulin response of the isolated, perfused rat pancreas. Inset: Integratedinsulin responses of the glucose control, hGIP and porcine GIP compiled from thesame data. All insulin responses were compared to the release associated withglucose alone.* p <0.05, hGIP n=7, porcine GIP n=8, glucose n=6.250*200EE* GlucoseI]] Human GIPPorcine GIP75002500Human GIP---0---- Glucose0 10 20 30 40Time (mm)74A 1 2 3 497—$-684329 f18ifIFigure 22: SDS-PAGE comparison of the induced expression of glutathione Stransferase and glutathione S-transferase/hGIPLeU 14 fusion proteins(A) Lane,1: molecular weight marker2: DH5a/pGEX 2T total cell protein3: DH5ct/pGEX 2T unbound fraction following a passage over aglutathione-Sepharose column4: DH5ct/pGEX 2T bound fraction following a passage over aglutathione-Sepharose columii; the open triangle indicates the putativeglutathione S-transferase(B) Lane,1: molecular weight marker2: DH5ct/pGEX 2T hGIPLeu total cell protein3: DH5cdpGEX 2T hGIPLU 14 unbound fraction following a passage overa glutathione-Sepharose column4: DH5a/pGEX 2T hGIPLeu 14 bound fraction following a passage over aglutathione-Sepharose column; the closed triangle indicates the putativeglutathione S-transferase hGIPLeU 1475of GSThGIPLeu 14 in E. coil DH5a cells was approximately 4.8 mg ofprotein/liter of bacterial culture.IVb) Optimization of GST-hGIPLCU 14 expression1) Expression Of GSThGIPLeu 14 as a function of bacterial strain:Fusion protein yields were examined in 7 strains of E. coil. Western blot analysisof the bacterial cell lysate following a 2 h induction period revealed thatrecombinant protein levels were approximately 2 to 3 fold higher in the Topp 3strain of E. coil (Figure 23A/B, lane 4).2’) Induction time of gene expression versus fusion protein yields:The possibility existed that the fusion protein yields may have been compromiseddue to enzymatic degradation in situ and, if so, that the degree of degradation wasa function of culture time following the onset of gene expression. Hence, fusionprotein yields were examined for induction times ranging from 30 mm to 24 h.Western blot analysis of the bacterial cell lysate following the various inductionperiods revealed that the recombinant protein levels peaked following a 4 hinduction period and declined marginally thereafter (Figure 24A!B, lane 5).Overall, the optimization strategy resulted in an approximately 3-fold increase inhGIPLeu 14 yields per liter of bacterial culture.IVc) CNBr cleavage of the GSThGIPLeu 14 fusion proteinThe translated fusion product was comprised of 2 domains: an amino tenninal GSTmoiety, a carboxy terminal hGIPLeU 14 moiety and an intervening methionine residue.Following the isolation of the fusion protein by affinity chromatography,76A1 297 —6843 —2918t14Figure 23: SDS-PAGE (A) and western blot (B) analysis of the influence of bacterialstrain on GSThGIPLeu 14 expression levels. Lane 1 is the molecular weight marker.Lanes 2 through 8 are total cell lysate samples from Topp 1, Topp 2, Topp 3, Topp 4,Topp 5, Topp 6 and DH5a strains of E. co/i. The triangle indicates theimniunoreactive fusion protein.3 4 5 6 7 8 8I77A 1 2 3 4 5 6 7 8 B 3 4 5 6 7 89768Figure 24: SDS-PAGE (A) and western blot (B) analysis of the influence ofinduction time on GST-hGIPLU 14 expression levels. Lane 1 is the molecular weightmarker. Lanes 2 through 8 are the total cell lysate samples following 30 mm and 1, 2,4, 8, 12 and 24 h induction periods.478CNBr cleavage of the intervening methionine residue was used to - release thehGIPLeu 14 domain. The reaction was apparently completed in 6 h as indicated bythe disappearance of the 29 kDa fusion protein band and associated appearance ofseveral bands ranging in size from approximately 1 kDa to 14 kDa (Figure 25A,lanes 2 and 3). These were consistent with the theoretical digestion products ofGSThGIPLeu 14 with CNBr (Table 4). Western blot analysis using the LMRanti-GIP polyclonal antibody, prepared in rabbit, confirmed that CNBr exposurereleased an inimunoreactive recombinant product that was the same molecularweight as porcine GIP (Figure 25B, lane 3). However, a second immunoreactiveproduct was noted with a molecular weight of approximately 11 kDa (Figure 25B,lane 3) suggesting that the reaction had not gone to completion. Thisiminunoreactive product was resistant to 28 h CNBr exposures and CNBr/substratemolar ratios in excess of 14,000 to 1.IVd) Purification of hGIPLeU from the CNBr digestion mixtureThe products of the CNBr digest were partially resolved on a C 18 reverse phasecolumn over a 28-35% gradient of acetonitrile in water containing 0.0 1% TFA.Passage over the C18 column resulted in a broad peak (Figure 26) which could not befurther resolved via a refinement to the buffer gradient. The recombinant hGIPLeu 14eluted in 32% acetonitrile (Figure 26) and comprised approximately 10% of the totalprotein in this fraction (Figure 26A!B, lane 4). Fractions containing theimmunoreactive material were resuspended in phosphate buffer, pH 6.0, and furtherresolved on a suiphapropyl column. The proteins were eluted with a 10 to 75 mMNaCl gradient in phosphate buffer. The resultant profile contained at least 10 peaks(Figure 27) which were collected, concentrated by lyophilization, and desalted by asecond passage over a C 18 column using a 0-50% gradient of acetonitrile in watercontaining 0.0 1% TFA. Suiphapropyl peak 5 was found to contain a near79A143 —i29181462Figure 25: SDS-PAGE (A) and western blot (B) analysis of the CNBr cleavage andpurification Of hGIPLeu 14 from the GSThGIPLeu 14 fusion protein.Lane,1: molecular weight marker2: uncleaved GST-hGIPLCU 143: GST-hGIPLU 14 following CNBr exposure; triangle in (B) indicates the11 kDa immunoreactive cleavage product4: partially-purified cleavage products following a passage over a C 18reverse phase column5: purified hGIPLeu 4 following a passage over an HPLC suiphapropylcolumn6: natural porcine GIP2 3 4 5 6’ B 3 4 5 64181462ij280Position in sequence Length (amino acids) Weight (kDa)ser2to met 69 68 8.2ala 70 to met 81 12 1.4leu 82 to met 94 13 1.4leu95tomet 129 35 4.0leu 130 to met 132 3 0.4phe 133 to met 154 22 2.7leu 155 to met 165 11 1.3asp 166 to met 168 3 0.4Cys 169 to met 225 50 5.7tyr 226 to gin 268 42 5.0(hGIPLeII 14)Table 4: Predicted proteolytic fragments generated following the exposure of GSThGIPLeuI4 to cyanogen bromide.81U2a>Figure 26: Reverse phase RPLC proffle of CNBr-digested GST-hGLP14resolved on a 28-35% gradient of acetonitrile in water containing 0.1% TFA. Thegradient ran from 9 to 19 mm on the chromatogram followed by a wash in 70%acetonitrile. The arrow indicates the region of the profile corresponding to theelution of recombinant hGIPj14.1MINUTES x 1082C>Figure 27: HPLC suiphapropyl profile of CNBr-digested GST-hGIPj resolvedon a 10-75 mM gradient of NaCI in 50 mM phosphoric acid, pH 6.0. The gradientran from 9 to 19 mm on the chromatograin followed by a wash in 0.5 M NaC1 in50 mM phosphoric acid, pH 6.0.0.58MINUTESxIO183homogeneous preparation of hGIPLeU 14 as evidenced by the HPLC profile (Figure28), SDS-PAGE analysis (Figure 25A, lane 5) and western blot analysis (Figure25B, lane 5). The yield of hGIPLeU 14 was approximately 100 jig/liter of bacterialculture.IVe) Amino acid sequence analysis of recombinant hGIPLeU 14The cleavage specificity of the cyanogen bromide was confirmed by gas-phaseNH2-terminal amino acid sequence analysis of the cleaved product. The first 13amino acids of the purified recombinant were identical to wild-type hGIP.IVf) Bioassay of recombinant hGIPLeU 14Both the recombinant hGIPLCU 14 and native porcine GIP were perfused throughthe isolated rat pancreas as a gradient from 0 to 1 ng/ml with a perfusateconcentration of 16.7 mM glucose (Figure 29). When the variability of the insulinresponses from individual experiments was taken into consideration, there was nosignificant difference between the insulin responses to the two peptides.V. ALANINE SCANNTNG MUTAGENESIS OF hGIPLeul4Alanine residues were substituted across the 19 to 30 domain of hGIPLeu 14 byPCR mutagenesis of the hGIPLeU 14 clone. Three individual substitutions, each 4alanine residues in length, resulted in the generation of GST-coupled fusionproteins corresponding to hGIPLeU 14, Ala 19-22’ hGIPLeu 14, Ala 23-26 and hGIPLeu 14,Ala 27-30’ The expression, purification and CNBr cleavage protocols of the alaninesubstituted, GST-coupled fusion proteins were identical to those used in thegeneration of hGIPLCU 14 (refer to sections IVa, b and c) except where noted.84I-.C;;- 4-2Figure 28: Reverse phase HPLC profile of suiphapropyl peak 5 (hGlli4)resolved on a 0-50% gradient of acetonitrile in water containing 0.1% TFA. Thegradient ran from 9 to 19 mm on the chromatogram followed by a wash in 70%acetonitrile.6-0•MINTJTES X 1018580-GlucoseGIPLeu 14Porcine GIP50Time (mm)Figure 29: Effect of a linear gradient of recombinant human GIP 14 or porcineGIP on the insulin response of the isolated perfused rat pancreas. Inset: Integratedinsulin responses of the glucose control, GIPj 14 and porcine GIP compiled fromthe same data. All insulin responses were compared to the release associated withglucose alone.* p <0.05, GIP14n4, porcine GIP n5, glucose n=6.3000*T6020-Glucose*__DII GIPLeu 14Porcine GIP1000D0 10 20 30 40086Va) Purification of GSThGIPLeu 14, Ala 23-26 from the transformed E. coil Topp 3cell lysate:Purification of GSThGIPLeu 14, Ala 23-26 by affinity chromatography resulted in thegeneration of a 95% pure preparation of inimunoreactive fusion protein asindicated in the SDS-polyaciylamide gel and associated western blot (Figure30A/B, lane 2). The yield of fusion protein was approximately 28 mg/liter ofbacterial culture.Vb) CNBr cleavage of the GSThGIPLeu 14, Ala 23-26 fusion protein:GSLhGIPLeu 14. Ala 23-26 , like GSThGIPLeu 14’ was partially resistant to CNBrexposure. As indicated by the disappearance of the 29 kDa fusion protein bandand associated appearance of several bands ranging in size from approximately 1kDa to 14 kDa, the reaction was apparently completed in 6 h (Figure 30A, lanes 2and 3). Western blot analysis using the LMR 34 anti-GIP polyclonal antibody,prepared in rabbit, confirmed that CNBr exposure released an immunoreactiverecombinant product that was the same molecular weight as porcine GIP (Figure30B, lane 3). However, a second immunoreactive product was noted with amolecular weight of approximately 11 kDa (Figure 30B, lane 3) suggesting that thereaction had not gone to completion. This immunoreactive product was resistantto 28 h CNBr exposures and CNBr/substrate molar ratios in excess of 14,000 to 1.Vc) Purification of hGIPLeu 14. Ala 23-26 following the CNBr digestion:The products of the CNBr digest were partially resolved on a C 18 reverse phasecolumn over a 28-35% gradient of acetonitrile in water containing 0.0 1% TFA.Passage over the C 18 column resulted in a broad peak (Figure 31) which could not befurther resolved via a refinement to the buffer gradient. The recombinant hGIPLCU 14,Ala 23-26 eluted in 33% acetonitrile (Figure 31). Fractions containing the874 5Figure 30: SDS-PAGE (A) and western blot (B) analysis of the CNBr cleavage andpurification of hGIPLeu 14. Ala 23-26 from the GST-fusion protein.Lane,1: molecular weight markers2: uncleaved GSThGIPLeu 14. Ala 23-263: GST-hGIPLU 14. Ala 23-26 following CNBr exposure; triangle in (B)indicates the imrnunoreactive 11 kDa cleavage product4: purified hGIPLeU 14, Ala 23-26 following passages over HPLC reversephase and suiphapropyl columns5: natural porcine GIP4 5A1 2 34329 .18146.2888C>40Figure 31: Reverse phase FfPLC proffle of CNBr-digested GSThGIPLeul4 Ala 23-26 resolved on a 28-35% gradient of acetonitrile in watercontaining 0.1% TFA. The gradient ran from 9 to 19 mm on the chromatogramfollowed by a wash in 70% acetonitrile. The arrow indicates the region of theprofile corresponding to the elution of recombinant hGIPii4 Ala 23-261MIN1JES x 1089inimunoreactive material were resuspended in phosphate buffer, pH 6.0, andfurther resolved on a suiphapropyl column. The proteins were eluted with a 10 to75 mM NaC1 gradient in phosphate buffer. The resultant profile contained at least7 peaks (Figure 32) which were collected, concentrated by lyophilization, anddesalted by a second passage over a C18 column using a 0-50% gradient ofacetonitrile in water containing 0.01% TFA. Suiphapropyl peak 4 was found tocontain a near homogeneous preparation of putative hGIPLeU 14, Ala 23-26 asevidenced by the HPLC profile (Figure 33), SDS-PAGE analysis (Figure 30A, lane4) and western blot analysis (Figure 30B, lane 4).Vd) Amino acid sequence analysis of recombinant hGIPLeU 14. Ala 23-26The cleavage specificity of the cyanogen bromide was confirmed by gas-phaseNH2-terminal amino acid sequence analysis of the cleaved product. The first 5amino acids of the purified recombinant were identical to wild-type hGIP.Ve) Bioassay of recombinant hGIPLCU 14. Ala 23-26The recombinant hGIPLeu 14, Ala 23-26 was perfused through the isolated ratpancreas as a gradient from 0 to 1 ng/ml with a perfusate concentration of 16.7mM glucose. The substitution of the wild-type amino acids corresponding toresidues 23 to 26 of GIP, with alanine, resulted in the loss of biological activitywhen compared to the insulin response to native porcine GIP (Figure 34).Vf) Purification of GSThGIPLeu 14. Ala 19-22 from the transformed E. coil Topp 3cell lysate:Purification of GSThGIPLeu 14. Ala 19-22 by affinity chromatography resulted in thegeneration of a 50% pure preparation of inmiunoreactive fusion protein as indicated inthe SDS-polyacrylamide gel and associated western blot (Figure 35A1B, lane 4).90Figure 32: HPLC suiphapropyl profile of CNBr-digested GST-hGIPj 14, 23-26resolved on a 10-75 mM gradient of NaC1 in 50 mM phosphoric acid, pH 6.0. Thegradient ran from 9 to 19 mm on the chromatogram followed by a wash in 0.5 MNaC1 in 50 mM phosphoric acid, pH 6.0.20-6MINUTESx1O’91S60>42Figure 33: Reverse phase HPLC profile of suiphapropyl peak 4(hGIPj 14, Ala 23-26) resolved on a 0-50% gradient of acetonitrile in watercontaining 0.1% TFA. The gradient ran from 9 to 19 mm on the chromatogramfollowed by a wash in 70% acetonitrile.MINTJThS x W292500040003000.z20001000•0Time (mm)0— GIP Leu 14, Ala 23-260 Porcine GIPFigure 34: Effect of a linear gradient of recombinant hwflafl GIP 14, Ala 23-26 orporcine GIP on the insulin response of the isolated perfused rat pancreas.Inset: Integrated insulin responses of the glucose control, GIP 14, Ma 23-26 andporcine GIP compiled from the same data. All insulin responses were compared tothe release associated with glucose alone.* p <0.05, hGIP 14, Ala 23-26 n=5, porcine GIP n=4, glucose n=6.-a- 80ao60a 4020coj GlucoseGIP Leu 14, Ala 23-26flj Porcine GIP0 10 20 30 40 50- - -- •- - -- Glucose93BA1 2 3 49729______18 *L14-Figure 35: SDS-PAGE (A) and western blot (B) analysis of the purification of GSThGIPLeu 14, Ala 19-22.Lane,1: molecular weight marker2: Topp 3/pGEX 2T hGIPLeU 14. Ala 19-22 total cell protein3: Topp 3/pGEX 2T hGIPLeu 14, Ala 19-22 unbound fraction following apassage over a glutathione-Sepharose column4: Topp 3/pGEX 2T hGIPLeu 14. Ala 19-22 bound fraction following apassage over a glutathione-Sepharose column, the arrow indicates theputative immunoreactive glutathione S-transferase hGIPLCU 14, Ala 19-2294The yield of fusion protein was approximately 4.3 mg/liter of bacterial culture.Vg) CNBr cleavage of the GSThGIPLeu 14, Ala 19-22 fusion protein:CNBr digestion of GSThGIPLeu 14, Ala 19-22 was performed under identicalconditions to GST-hGIP 14 and GST-hGIP 14, Ala 23-26 The digest resulted inthe disappearance of the 29 kDa fusion protein band and associated appearance ofseveral bands ranging in size from approximately 1 kDa to 14 kDa (Figure 36A,lanes 2 and 3). Western blot analysis using the LMR 34 anti-GIP polyclonalantibody, prepared in rabbit, confirmed that CNBr exposure released animmunoreactive recombinant product that was the same molecular weight asporcine GIP (Figure 36B, lane 3) but in diminished yields when compared to theother fusion products. As before, a second immunoreactive product was notedwith a molecular weight of approximately 11 kDa (Figure 36B, lane 3) suggestingthat the reaction had not gone to completion. The yield of GSThGIPLCU 14, Ala 19-22 was not affected by an extension of the CNBr exposure timeor the use of CNBr/substrate molar ratios in excess of 14,000 to 1. The CNBrdigestion products were partially resolved on a C18 reverse phase column over a28-35% gradient of acetonitrile in water containing 0.01% TFA. Passage over theC18 column resulted in a broad peak (Figure 37) which could not be furtherresolved via a refinement to the buffer gradient. HGIPLeu 14, Ala 19-22 eluted inapproximately 35% acetonitrile. However, the yield of iminunoreactive product ofthe correct predicted molecular weight was negligible.95Al43-2918 ,14 .6.2.22 3 418146.22.92 3I4Figure 36: SDS-PAGE (A) and western blot (B) analysis of the CNBr cleavage ofhGIPLeu 14, Ala 19-22Lane,1: molecular weight marker2: uncleaved GSThGIPLeu 14, Ala 19-223: GSLhGIPLeu 14. Ala 19-22 following CNBr exposure; triangle in (B)indicates the imniunoreactive 11 kDa cleavage product4: natural porcine GIP2996200>100Figure 37: Reverse phase HPLC proffle of CNBr-digested GSThGIPJRu14 Ala 19-22 resolved on a 28-35% gradient of acetonitrile in watercontaining 0.1% TFA. The gradient ran from 9 to 19 mm on the chromatogramfollowed by a wash in 70% acetomtrile The arrow indicates the region of theproffle corresponding to the elution of recombinant hGIPji4 Ala 19-22JrM1NTJTESxlO397Vh) Purification of GSThGIPLeu 14. Ala 27-30 from the transformed E. o1i Topp 3cell lysate:Purification of GSThGIPLeu 14, Ala 27-30 by affinity chromatography resulted in thegeneration of a 80% pure preparation of immunoreactive fusion protein asindicated in the SDS-polyacrylamide gel and associated western blot (Figure38A/B, lane 3). The yield of fusion protein was approximately 14 mg/liter ofbacterial culture.Vi) CNBr cleavage of the GSThGIPLeu 14, Ala 27-30 fusion protein:CNBr digestion of GST-hGIPLCU 14. Ala 27-30 was performed under identicalconditions to the other recombinant fusion proteins. The digest resulted in thedisappearance of the 29 kDa fusion protein band and associated appearance of severalbands ranging in size from approximately 1 kDa to 14 kDa (Figure 39A, lanes 2 and3). Western blot analysis using the LMR 34 anti-GIP polyclonal antibody, preparedin rabbit, confirmed that CNBr exposure released an immunoreactive recombinantproduct that was the same molecular weight as porcine GIP (Figure 39B, lanes 2 and3). As before, a second immunoreactive product was noted with a molecular weightof approximately ii kDa (Figure 39B, lane 2) suggesting that the reaction had notgone to completion. The yield of GSThGIPLeu 14, Ala 27-30 was not affected by anextension of the CNBr exposure time or the use of CNBr/substrate molar ratios inexcess of 14,000 to 1. Like GSThGIPLeu 14, Ala 19-22’ one or more of the methionineresidues in GSThGIPLeu 14. Ala 27-30 appeared to be significantly resistant to CNBrexposure. Nonetheless, the CNBr digestion products were partially resolved on a C18reverse phase column over a 28-35% gradient of acetonitrile in water containing0.0 1% TFA. Passage over the C18 column resulted in a broad peak (Figure 40) whichcould not be further resolved via a refinement to the buffer gradient. HGIPLeu 14, Ala27-30 eluted in approximately 34-35% acetonitrile. However, like hGIPLeu 14. Ala 19-22’98A1 2 3 B 1 2 397__p 9768 . 6843 432929_____18 1814 14Figure 38: SDS-PAGE (A) and western blot (B) analysis of the purification of GSThGIPLeu 14. Ala 27-30Lane,1: molecular weight marker2: Topp 3/pGEX 2T hGIPLeu 14, Ala 27-30 unbound fraction following apassage over a glutathione-Sepharose column3: Topp 3/pGEX 2T hGIPLeU 14, Ala 27-30 bound fraction following apassage over a glutathione-Sepharose column; the arrow indicates theputative imniunoreactive glutathione S-transferase hGIPLeu 14, Ala 27-3099B 1 2A 1 2 3 343 4329 2918 1814 1146.22.9 2.9Figure 39: SDS-PAGE (A) and western blot (B) analysis of the CNBr cleavage ofhGIPLeu 14. Ala 27-30Lane,1: molecular weight marker2: GSThGIPLCU 14, Ala 27-30 following CnBr exposure; triangle in (B)indicates the immunoreactive 11 kDa cleavage product3: natural porcine GIP10010C5Figure 40: Reverse phase 1{PLC profile of CNBr-digested GSThGIPLeu 14, Ala 27-30 resolved on a 28-35% gradient of acetonitrile in watercontaining 0.1% TFA. The gradient ran from 9 to 19 miii on the chromatograrnfollowed by a wash in 70% acetonitrile. The arrow indicates the region of theprofile corresponding to the elution of recombinant hGIPi4 Ma 27-30MINUTES x 101101the yield of immunoreactive product of the correct predicted molecular-weight wasnegligible.VI. ALTERNATIVE CLEAVAGE AND PURIFICATION STRATEGY FORGSThGIPLeu 14. Ala 19-22 AND GSLhGIPLeu 14. Ala 27-30Given the low yields of the fusion protein digests and subsequent purification ofhGIPLeu 14. Ala 19-22 and hGIPLeu 14, Ala 27-30’ an alternative strategy was devisedwhich took advantage of a pre-engineered thrombin-recognition site encodeddirectly upstream of the polylinker. The product of a thrombin digest of the GSTfusion constructs was composed of a short amino terminus glycine-serinemethionine (gsm) peptide linked to the GIP analog and therefore will be referred toas “gsm-hGIP”.VIa) Thrombin digest of GST-hGIPj 14. Ala 19-22’ GSThGIPLeu 14. Ala 23-26-flGSThGIPLeu 14, Ala 2730:GSThGIPLeu 14. Ala 19-22’ a GSLhGIPLeu 14, Ala 23-26 control and GSThGIPLeu 14, Ala27-30 were digested with thrombin for 3 h at 3 7°C. The digestion of GSThGIPLeu 14,Ala 23-26 resulted in the appearance of a 4 kDa band, and virtual disappearance of a 29kDa band on an SDS-polyacrylamide gel (Figure 41, lanes 2 and 4) suggesting that thedigestion had gone to near completion. The digestion of GSThGIPLeu 14, Ala 19-22 andGSThGIPLeu 14, Ala 27-30’ under identical conditions, resulted in only the partialcleavage of the fusion constructs as evidenced by the limitedappearance/disappearance of the associated 4 kDa and 29 kDa bands on the SDSpolyacrylamide gel (Figure 41, lanes 2, 3 and 5). However, the latter two digestsresulted in the generation of a significantly higher amount of precipitate.102Figure 41: SDS-PAGE analysis of the thrombin-digested, alanine-substituted mutants.Lane,1: molecular weight marker2: uncut fusion protein (GSThGIPLeu 14. Ala 23-26)3: GSThGIPLeu 14. Ala 19-22 supernatant following a 3 h thrombin digest4: GSThGIPLeu 14, Ala 23-26 supernatant following a 3 h thrombin digest5: GSThGIPLCU 14, Ala 27-30 supernatant following a 3 h thrombin digest6: urea-extracted GSThGIPi 14, Ala 19-22 precipitate following a 3 hthrombin digest7: urea-extracted GSThGIPLeu 14. Ala 27-30 precipitate following a 3 hthrombin digest8: natural porcine GIP1 2 34 56 78103This precipitate contained the majority of the GST-hGIP as indicated by anexamination of the urea extracted products of the precipitate by SDS-PAGEanalysis (Figure 41, lanes 6 and 7). Nonetheless, the presence of the predominant29 kDa band suggested that only limited proteolysis had occurred (Figure 41, lanes6 and 7).VIb) HPLC analysis of thrombin-digested GSThGIPLeu 14, Ala 19-22’ GSThGIP.14, Ala 23-26 and GSThGIPLeu 14, Ala 27-30:-Each of the thrombin digestion products of the GST-fused, alanine-substitutedmutants was resolved on a C18 reverse phase column over a 28-35% (GSThGIPLeu 14. Ala 23-26) or 3 3-50% (GSThGIPLeu 14, Ala 19-22 and GSThGIPLeu 14. Ala27-30) gradient of acetonitrile in water containing 0.01% TFA. Novel peaks weregenerated following each of the digests which eluted at 10.87, 13.20 and 10.47mm for the digests of GSThGIPLeu 14, Ala 19-22’ GSLhGIPLeu 14. Ala 23-26 andGSThGIPLCU 14, Ala 27-30 respectively (Figure 42B, D and F). SDS-PAGE andwestern blot analysis indicated that each of the novel peaks corresponded to aninimunoreactive product of the correct predicted molecular weight. However,based upon the peak area, the yield of gsm-hGIP 14, Ala 23-26 was at least onehundred-fold higher than that of the other two mutants (Figure 42B, D and F).VIc) Optimization of the thrombin cleavage reaction:Several modifications of the digestion protocol were examined in an attempt tooptimize the yields of gsm-hGIP 14, Ala 19-22 and gsm-hGIP 14, Ala 27-30(Table 5). Protein yields were not increased significantly as a function of thedigestion protocol. The yields of gsm-hGIP11 14, Ala 19-22 and gsm-hGIP 14, Ala27-30 were each approximately 790 ng/mg of fusion protein.104BFigure 42: Reverse phase HPLC profiles of GSThGIPLeu 14, Ala 19-22’ GSThGIPLeu14, Ala 23-26 and GSThGIPLCU14 Ala 27-30 prior to thrombin exposure (A, C and Erespectively) and following a thrombin digest (B, D and F respectively). The peptidesassociated with chromatograms A, B, E and F were resolved on a 3 3-40% gradient ofacetonitrile in water containing 0.1% TFA. The peptides associated withchromatograms C and D were resolved on a 28-35% gradient of acetonitrile in watercontaining 0.1% TFA. The gradient ran from 9 to 19 mm on each of thechromatograms followed by a wash in 70% acetonitrile.AI—C1202’E0MINUTES X l0 MINUTESX 10_i105Parameter: Conditions:Time Incubation at 37°C for 2, 4, 8 and 18 h.Enzyme concentration 6 to 600 units of thrombinlmg fusionprotein added at time 0 of the digest.Enzyme delivery 6 units of thrombinlmg fusion proteinadded to the reaction mixture at time 0and every 2 h thereafter for 8 h.Detergent concentration Digestion buffer supplemented with 0,0.0075, 0.01, 0.0 15, 0.02 and 0.025 %SDS.pH Digestion reaction buffered at pH 5.5,6.0, 6.5, 7.2, 7.6 and8.0.Temperature Digestion in buffer at 37°, 39°, 41° and43°C.Table 5: Digestion conditions for the cleavage of gsm-hGIP 14. Ala 19-22 and gsmhGIPLeu 14. Ala 27-30106VId) Cyanogen bromide digestion of gsm-hGIP 14. Ala 19-22 gsm.-hGIP 14.Ala 23-26 and gsm-hGIP 14, Ala 27-30.Sixteen micrograms of gsm-hGIP 14, Ala 19-22 and gsm-hGIP 14, Ala 27-30 werepurified from approximately 20 mg of the associated GST-fusion protein. Inseparate reactions, this material, in addition to 170 jig of gsm-hGIP 14, Ala 23-26control, was digested with CNBr for 6 h at room temperature and concentrated in aSpeed-Vac rotary evaporator. The digestion mixtures were resolved on a C18reverse phase column over either a 28-35% (gsm-hGIP 14, Ala 23-26) or 28-40 %and 33-40 % (gsrn-hGIP 14. Ala 19-22 and gsm-hGIP 14, Ala 27-30 respectively)gradient of acetonitrile in water containing 0.0 1% TFA:VIe) Reverse phase purification of cyanogen bromide-treated gsm-hGIP 14, Ala23-26.Passage of CNBr-treated gsm-hGIP 14, Ala 23-26 over a Cl 8 column resulted inthe resolution of 5 peaks (Figure 43B). Four peaks which eluted at novel retentiontimes (Figure 43B, peaks 1, 2, 3 and 5) and one which co-eluted with theuncleaved gsm-hGIP 14, Ala 23-26 (Figure 43A, B, and C). All five peakscontained inimunoreactive peptides of similar molecular weight as indicated bySDS-PAGE and western blot analysis (Figure 44A, B). Peak 2 was retained forfurther analysis.1) Amino acid sequence analysis of recombinant hGIPLeu 14. Ala 23-26 peak 2:The cleavage specificity of the cyanogen bromide was confinned by gas phaseNH2-terminal amino acid sequence analysis of the cleaved product. The first 5amino acids of the purified recombinant were identical to wild-type hGIP.107AFigure 43: Reverse phase HPLC profiles of gsm-hGIP 14, Ala 23-26 prior to (A), andfollowing (B), CNBr exposure. Each was resolved on a 28-35% gradient ofacetonitrile in water containing 0.1% TFA which ran from 9 to 19 mm on thechromatograms. An overlay of the profiles (C) shows the co-elution of gsm-hGIP14, Ala 23-26 (A) and peak 4 (B).MTNTJThSX 1OB0>to.-t02—0—-J0>I 1 IMINUTES X 10MiNUTES X 1O108Figure 44: SDS-PAGE (A) and western blot (B) analysis of CNBr-digested gsmhGIPLeu 14, Ala 23-26• Lane 1 is the molecular weight markers. Lanes 2 through 7correspond to HPLC peaks 1 to 5 (figure 43B). Lane 8 is natural porcine GIP.1092) Bioassay of recombinant hGIPL 14. Ala 23-26 peak 2:The recombinant hGIPLeu 14, Ala 23-26 peak 2 was perfused through the isolated ratpancreas as a gradient from 0 to 1 ng!ml with a perfusate concentration of 16.7mM glucose. HGIPLeu 14, Ala 23-26 peak 2 was not insulinotropic when comparedto the insulin response to native porcine GIP (Figure 45)VIf) Reverse phase purification of cyanogen bromide-treated gsm-hGIP 14, Ala19-22 and gsm-hGlP 14, Ala 27-30.Passage of the 16 pg aliquots of CNBr-treated gsm-hGIP 14, Ala 19-22 or gsmhGIPLeu 14, Ala 27-30 over a C18 reverse-phase column using a 28-40% (hGIPLCU 14,Ala 19-22) or 33-40% (hGIPLeu 14. Ala 27-30) gradient of acetonitrile in watercontaining 0.0 1% TFA resulted in the generation of poorly-defined peaks (Figure46). Due to insufficient yields, protein sequence analysis and biological analysiscould not be performed with this material. The experimental results associatedwith all of the recombinant GIP analogues and synthetic/proteolytic GIP fragmentsare summarized in Table 6.110Time (mm)—D—— GIP Leu 14, Ala 23-26 Peak 2PorcineGIP- --- •- - -- GlucoseFigure 45: Effect of a linear gradient of recombinant human GIPLeU 14, Ala 2 3-26peak 2 or porcine GIP on the insulin response of the isolated perfused rat pancreas.Inset: Integrated insulin responses of the glucose control, GIP 14, Ala 23-26 peak2 and porcine GIP compiled from the same data. All insulin responses werecompared to the release associated with glucose alone.* p <0.05, GIPj 14, Ala 23-26 peak 2 n=3, porcine GIP n=r3, glucose n=3.E1 GlucoseGIP Leu 14, Ala 23-26 Peak 2Jjj Porcine GIP0 10 20 30 40 50111I’S-Figure 46: Reverse phase HPLC profiles of gsm-hGIP 14, Ala 19-22 and gsmhGIP114 Ala 2730 prior to CNBr exposure (A and C respectively) and followingCNBr cleavage (B and D respectively). The gsm-proteins were resolved on a 33-40 %gradient of acetonitrile in water while the CNBr-treatedgsm-hGIP 14. Ala 19-22 and gsm-hGIP14 Ala 27-30 were resolved on 28-40% and33-40% gradients respectively.-,C-JC>MINUTES X 10.1 MINUTES X 100.0.4MiNurEs x io MINUTES X 10.1112II Peptide N-terminal sequence Insulinotropic activity I!native porcine GIP yes - full length sequence +++++obtained by otherssynthetic G1P1530 yes-peak 3 derived from the no ++enterokinase digest ofG1P15..30synthetic G1P1730 yes ++G1P19..30 yes - 1 1 amino acids +recombinant hGIP yes - 29 amino acids +++++recombinant hGIPjeii yes - 13 amino acids +++++recombinant yes - 5 amino acids-hGIP11 14, Ala 23-26recombinant yes - 5 amino acids-hGIPLeu 14. Ala 23-26peak 2recombinant no unknownhGIPieii 14. Ala 19-22recombinant no unknownhGIPLe11 14 Ala 27-30Table 6: Summary of experimental data113DISCUSSIONI. FRAGMENT ANALYSIS OF PORCINE GIPPeptide fragment generation and analysis have been used to investigate thestructure/function relationship between GIP and pancreatic insulin release. Bothsynthetic G1P130 (Pederson et al, 1990) and proteolytically-derived G1P1542(Pederson and Brown, 1976) potentiated insulin release fiom the isolated, perfusedrat pancreas which implied that the bioactive domain of the molecule lies in theshared 15-30 sequence. Nonetheless, synthetic G1P1530 was not bioactive (RAPederson, unpublished), suggesting that the deletion of the flanking residuesdisrupted the integrity of the molecule such that its biological activity wasimpeded. However, GIP residues 15 and 16 are aspartic acid and lysinerespectively. Therefore it was hypothesized that the removal of the 1 to 14 domainof the molecule and concurrent exposure of these two charged residues may havepromoted an interaction between the NH2-terminus aspartic acidllysine residuesand an internal domain of the 15 to 30 fragment; an ionic interaction between theexposed charged residues and an internal charged domain may have resulted in thegeneration of a hairpin loop which sterically blocked or disrupted the amino acidsresponsible for the insulin response at the beta cell. If so, this may have beenresponsible for the abolition of insulinotropic activity seen in the perfusionexperiments. Therefore it followed that the proteolytic removal of the asparticacid!lysine dipeptide, with enterokinase, from synthetic G1P15..30 (to generateG1P17..30) might result in the restoration of biological activity by effectivelyunfolding the fragment and exposing the relevant amino acids.114The enterokinase digest of G1P15.30 resulted in the generation of 9 peaks on anHPLC reverse phase profile; two peaks which co-eluted with enterokinase andundigested G1P15..30 and seven peaks which eluted at novel retention times (Figure11), The generation of multiple peaks suggested that the specificity or the purityof the enterokinase preparation was suspect. The purest preparation of Sigmaenterokinase contains less than 1% free trypsin and less than 0.5% aminopeptidaseactivity; these contaminants were probably responsible for the generation of thedisparate peaks.In order to determine if the enterokinase treatment restored the biological activityof the G1P1530 fragment, HPLC peaks 3 through 8 (Figure 1 1C) were perfusedthrough the isolated rat pancreas.. Only peak 3 was found to stimulate insulinrelease significantly (Figure 13). Given that, a) G1P1530 is a relatively smallpeptide fragment whose bioactivity would be sensitive to extensive changes inprimary structure, b) the removal of the aspartic acidllysine residues represented arelatively minor disruption when compared to further fractionation of the peptideand, c) the peaks which were not bioactive likely represented relatively extensivecleavages of the synthetic 15 to 30 fragment, it was theorized that peak 3 wascomprised of G1P1730. However, the requirement of large amounts of the G1P15..30 starting material and associated low yields of peak 3 following the enterokinasedigest complicated the isolation of adequate amounts of this material required forprotein sequence analysis. Furthermore, maximal yields of peak number 3 wereobtained following a 60 mm digest and declined rapidly thereafter suggesting thatit was an intermediate of the proteolytic reaction. Hence a preparation of syntheticG1P1730 was obtained and perfused through the isolated rat pancreas. SyntheticG1P1730 and the enterokinase-derived peak 3 preparation potentiated insulin115release in a similar manner. Therefore the removal of the aspartic--acidllysineresidues from the biologically-inert G1P1530 peptide partially rescued theinsulinotropic activity of the fragment; the perfusion of a gradient of 0-1 ng/ml ofsynthetic G1P17.30 and HPLC peak 3 resulted in the restoration of 31% and 30%(respectively) of the insulinotropic activity when compared to the native full-length molecule. This supported the hypothesis that these two charged residueswere interacting with another domain of the peptide resulting in a conformationalchange which effectively shielded the biologically relevant amino acids.Interestingly, the removal of the first two amino terminal residues from full-lengthG1P142 results in the generation of biologically-inactive G1P342 (Brown et al,1981, Schmidt et al, 1986); presumably the NH2-tenninal amino tyrosine andalanine residues were required for biological activity. However this wasinconsistent with the observation that G1P1542 was partially insulinotropic(Pederson and Brown, 1976). In addition, the deletion of the glutamic acid residuein position number 3 to generate G1P442 restored the receptor-binding capacity ofthe molecule (Maletti et a!, 1986). This suggests that the proteolytically-exposedglutamic acid residue in position number 3, like the aspartic acidllysine residues inpositions 15 and 16, may be interacting with the molecule and interfering withbiological activity.G1P17..30 represented the smallest bioactive fragment of GIP generated to date. Inorder to define further the insulinotropic domain of the molecule, GIP 15-30 wasdigested with bovine tiypsin (which theoretically cleaves the peptide at arginine11618) to yield G1P1930. HPLC reverse phase analysis indicated that tryptic exposureresulted in the complete abolition of the G1P1530 peak and concurrent generationof two novel peaks eluting at 14.55 and 17.43 mill (Figure 12B, C). Gas phaseprotein sequence analysis of the peak eluting at 14.55 mm confirmed thespecificity of the tryptic digest to arginine 18 of GIP1530; the confirmed sequenceof the peptide associated with this peak corresponded to residues 19 to 29 of thefull-length molecule. An allocation for the residue corresponding to GIP residue30 was not possible. This is typical since the identification of the terminal residuein a sequence analysis is often difficult or impossible (personal communicationwith Sandie Kielland, University of Victoria Protein Microchemistry Center). Theremoval of residues 15 through 18 (that is, the generation of GIP19-30) resulted in apartial restoration of insulinotropic activity from the inactive 15 to 30 fragment.However, given the lower molecular weight of the GIP fragments when comparedto the full-length molecule, all of the fragment perflisions were performed with anapproximately 4-fold higher concentration of peptide when compared on a molarbasis. Nonetheless, the observation that equimolar perfusions of synthetic G1P15.30 versus G1P17..30 and G1P1930 resulted in significantly different insulin responsessupported the hypothesis that the amino terminus aspartic acidllysine residues ofthe G1P1530 fragment were interacting with another domain of the peptide. Thiswas presumably the result of a conformational change which effectively shieldedthe biologically relevant amino acids. However, both G1P17..30 and G1P1930displayed only a fraction of the insulinotropic activity of the full-length moleculedespite the higher concentration of peptide used in the perfusion experiment. Thiswas perhaps due to the loss of the stabilizing influence of the neighboring aminoacids and their associated contribution to the structural integrity of the molecule.Alternatively, the G1P1730 or G1P1930 fragments may have been preferentially117degraded in the pancreas preparation when compared to full-length GIP.Nonetheless, the retention of the partial biological activity of G1P17..30 and G1P19..30 coupled with the observations that, a) position 18 is not conserved betweenporcine and human GIP with no detriment to biological activity and, b) theisoleucine residue in position 17 is not conserved between GIP and the otherinsulinotropic members of the glucagon superfamily of polypeptides (Table 1),strongly suggests that residues 17 and 18 do not contribute to the insulin responseand that residues 19 to 30 solely comprise the bioactive domain of GIP.II. PROKARYOTIC EXPRESSION OF GIPDue to the observed decrease in bioactivity of the GIP fragments as a function ofpeptide size, a further resolution of the insulinotropic domain of GIP was notfeasible using fragment analysis. However, the molecular dissection of GIP wasstill attainable using molecular biological techniques; prokaryotic expression ofthe molecule would allow for the generation of novel, full-length isofonnscontaining defined substitutions within the 19 to 30 domain. This would minimizethe possibility that alterations in bioactivity were the result of a secondarydisruption in the structural integrity of the molecule and maximize the possibilitythat any deviations in insulinotropic activity were the result of the replacement ofamino acids directly involved in the insulin response.a) Expression of human GIP as a protein A fusion proteinGiven that prokaryotic and eukaryotic cells utilize different signals for theinitiation of transcription and translation, low protein yields are often observedwhen eukaryotic genes are expressed in prokaiyotic systems (Matsudaira, 1987).To address this problem, and to simplify the purification process, a protein A-hGIPfusion construct was expressed in E. coil. The design of the synthetic GIP gene118was based on the published cDNA sequence of hGIP (Takeda et al, 1987) and onthe codon usage of highly-expressed genes in E. coil (Maruyama, 1986).A synthetic hGIP gene, with a 5-prime sequence encoding the Aa-fibrinopeptidethrombin-recognition sequence (Blomback, 1969), was linked to a Staphylococcusaureus protein A gene which had been previously engineered into the pRIT 2Texpression vector. Expression of the chimeric gene resulted in the generation of afusion protein comprised of three domains: protein A, hGIP and an interveningthrombin-recognition sequence. Following the purification of the fusion proteinusing IgG-Sepharose affinity chromatography, the hGIP moiety was released viathrombin cleavage of the Aa-fibrinopeptide sequence. A second passage over theIgG-Sepharose column and subsequent gel exclusion chromatography stepremoved the thrombin and the majority of the non-specific binding proteins. Thepurified recombinant product was identified as hGIP by various methods usingnative porcine GIP as the standard; SDS-PAGE analysis indicated that therecombinant product was approximately the same molecular weight as porcine GIP(Figure 17B, lanes 5 and 6), retention times on a HPLC reverse phase columnindicated that the recombinant hGIP and porcine GIP demonstrated similarhydrophobicities (Figure 19A, B), the immunoreactivity of the recombinant wasconfirmed by ELISA using an anti-porcine GIP antibody (Figure 20) and peptideamino acid analysis verified the specificity of the thrombin digest and confirmedthe identity of the first 29 amino acids (Table 3).Although the preliminary yields were adequate, several concerns prompted a reexamination of the practicability of this system as a model for further mutationalanalysis of the insulinotropic domain of GIP:1191) theoretically, passage of the cell lysate over an IgG-Sepharose column shouldresult in the preferential isolation of the fusion protein given the strong interactionbetween protein A and the constant region of immunoglobins. In practicehowever, the purified fusion protein was heavily contaminated with non-specificbinding proteins (Figure 17, lane 7). This was probably due, in part, to thediversity of the class G immunoglobins that had been raised in the source animaland used in the generation of the commercial IgG-Sepharose preparation; thevariable regions of the inimunoglobins likely interacted with the prokaryoticproteins and thus contributed to the isolation of the contaminants. Although thesewere largely removed in subsequent purification steps, the gel exclusionchromatography step was difficult to reproduce. Given that subsequent workwould entail the generation of GIP isoforms, and that each would probably requirethe development of a novel purification protocol, the presence of significantamounts of contaminating protein would make future extractions difficult.2) The presence of protein A effectively nullified the use of immunogenic methodsto either quantify or identify the recombinant GIP (except the final and purestpreparation of the recombinant) since the presence of trace amounts of protein Acould be detected by ELISA or RIA analysis.3) The yield of recombinant hGIP was approximately 100 jig/liter of bacterialculture. Other expression systems were available which reported significantlyhigher yields. Given the inherent difficulty in isolating native GIP by classicalmeans, there was an impetus to develop a superior system for the generation ofrecombinant biological material. Therefore a second prokaiyotic expressionsystem was developed which benefited from the advantages affiliated with120prokaryotic fusion protein systems but lacked the difficulties associated with theuse of protein A.b) Expression of human GIP as a glutathione S-transferase fusion proteinA synthetic hGIP gene, utilizing the same codons as before but lacking thethrombin recognition sequence, was inserted into the pGEX 2T vector. PGEX 2Texpresses glutathione S-transferase and a thrombin recognition sequence upstreamof the polycloning site. The polycloning site is comprised of Barn HI, Sma I andEco RI restriction sites. Since these sites lie downstream of the codons which willeventually encode the thrombin cleavage site of the translated fusion construct, arecombinant insert will contain the NH2-terminus amino acid(s) encoded by therestriction site at the point of insertion. For example, if the synthetic hGIP genewas engineered with a 5-prime Barn HI site and 3-prime Eco RI site, the 5-primeBarn HI (GGATCC) site would encode glycine and serine residues at the aminoterminus of the recombinant GIP product following its release from the GSTmoiety by thrombin.Given that the deletion of the first two NH2-terminus amino acids of GIP resultedin the complete uncoupling of insulinotropic activity (Brown et al, 1981 Schmidtet al, 1986), GIP appears to be sensitive to deletions at its amino terminus andmight be equally sensitive to the addition of other residues. Therefore, amethionine codon was included directly upstream of the hGIP gene to facilitate theremoval of the glycine and serine residues and to supersede the requirement for athrombin digest. A methionine to leucine substitution was made at position 14 ofthe polypeptide such that it would remain resistant to CNBr exposure. Thissubstitution had been made previously with no detriment to biological activity(Horn et al, 1983).121The synthetic methionine-hGIPLeU 14 gene was inserted into the pGEX 2Texpression vector. Expression of the fusion protein was driven by the IPTGinducible Ptac promotor. The addition of IPTG to an E. co/i culture which hadbeen previously transformed with pGEX 2ThGIPLeu 14 resulted in the expressionof a 29 kDa GSThGIPLeu fusion protein. The fusion protein was purified usingaffinity chromatography by taking advantage of the strong interaction between theglutathione S-transferase moiety of the fusion protein and glutathione-Sepharose.Unlike the IgG-Sepharose affinity column used in the protein A fusion system,exposure of the total cell lysate to glutathione-Sepharose resulted in thepreferential binding and purification of the GSThGIPLeu The GSThGIPLeu 14preparation contained only one major contaminant as indicated by SDS-PAGEanalysis; a 25 kDa non-imniunoreactive band which comprised approximately30% of the total protein in the sample (Figure 25A, lane 2).The yield of fusion protein was 4.8 mg/liter of E. coil DH5cL culture. However anoptimization strategy was devised which resulted in an approximately 3-foldincrease in fusion protein yields. The fusion protein was digested with CNBr andthe recombinant hGIPLeU 14 was recovered by successive passages over C18reverse phase and suiphapropyl columns. As before, the identity of therecombinant hGIPLeu 14 was confirmed by various methods using native porcineGIP as the standard; SDS-PAGE analysis indicated that the recombinant productwas approximately the same molecular weight as porcine GIP (Figure 25A, lanes 5and 6), the immunoreactivity of the recombinant was confirmed by western blotanalysis using an anti-porcine GIP antibody (Figure 25B, lane 5) and amino acidsequence analysis verified the specificity of the CNBr digest and confirmed theidentity of the first 13 amino acids. There was no significant difference in the122insulin responses between recombinant hGIPj 14 and native porcine GIP (Figure29). A comparison of the reverse phase profiles of the purified hGIP derived fromthe protein A system and the GST system (Figures 19B and 28 respectively)revealed that the “GST-derived” recombinant hGIP was of a higher purity than the“protein A” derived hGIP; given the higher yields and purity of the hGIPLeU 14’ thepGEX 2T system was chosen for the mutational analysis of the insulinotropicdomain of GIP.III. SITE-DIRECTED MUTAGENESIS OF hGIPLeu 14A PCR mutagenesis protocol developed by Nelson and Long (1989) was used togenerate three hGIPLeU 14 mutants from a pUC l9hGIPLeu 14 template. Each wascomprised of a full-length hGIPLeU 14 molecule containing an alanine “window”which partially spanned the 19 to 30 domain of the polypeptide. The alaninesubstituted mutants were subcloned into the pGEX 2T vector and were expressedand purified under similar conditions to GSThGIPLeu 14; three individualsubstitutions, each 4 alanine residues in length, resulted in the generation of GSTcoupled fusion proteins corresponding to hGIPLeU 14, Ala 19-22’ hGIPLeu 14, Ala 23-26and hGIPLeu 14. Ala 27-30IV. CLEAVAGE AND ANALYSIS OF THE GSThGIPLeu MUTANTSFollowing the isolation of the fusion proteins by affinity chromatography, eachwas exposed to CNBr at a molar ratio of CNBr to peptide of 100:1 for 6 h at roomtemperature. One methionine residue appeared to be resistant to CNBr exposurein all of the GST-coupled fusion proteins, including GST-hGIPLU 14• The resultwas a spurious immunoreactive 11 kDa band, indicated by western blot analysis(Figures 25B: lane 3, 30B: lane 3, Figure 36B: lane 3, 39B: lane 2), which wasresistant to CNBr exposures using molar ratios of CNBr to protein exceeding12314,000:1 and reaction times in excess of 28 h. Initially this was assumed to be theresult of methionine sulfoxide formation at the methionine of interest since thiswould render it resistant to CNBr exposure (Gross, 1967). However, thepreferential formation of one methionine sulfoxide residue above the remaining 9methionine residues in the fusion construct seemed improbable. Nonetheless,given the adequate yields obtained from the hGIPLeU 14 clone and the high yieldsof fusion protein obtained from each of the alanine-substituted mutants, thedigestion and purification protocol was pursued.The development of the purification protocol for hGIPLeu 14, Ala 23-26 wasrelatively straightforward since this mutant and hGIPLeU 14 have an identicalisoelectric constant and similar hydrophobic properties. A virtually homogeneouspreparation of hGIPLeu 14. Ala 23-26 was prepared as indicated by reverse phaseHPLC (Figure 33), SDS-PAGE (Figure 30A, lane 4) and western blot analysis(Figure 30B, lane 4). Amino acid sequence analysis verified the specificity of theCNBr digest at the amino terminus and confirmed the identity of the first 5 aminoacids. The recombinant hGIPLeu 14. Ala 23-26 was perfused through the isolated ratpancreas as a gradient from 0 to 1 nglml with a perfusate concentration of 16.7mM glucose. The substitution of the residues corresponding to the 23-26 domainof hGIP (Val-Asn-Trp-Leu), with alanine, resulted in the virtual elimination ofinsulinotropic activity when compared to porcine GIP and hGIPLCU 14 controls.This suggests that one or more of the amino acids within this domain is involved ina critical interaction with the receptor.Notwithstanding the successful cleavage and purification of hGIP 14 andhGIPLeu 14. 23-26’ the cleavage and isolation of the remaining two alaninesubstituted mutants proved to be far more difficult. Specifically, the yields of124hGTPLeu 14, Ala 19-22 or hGIPLeu 14. Ala 27-30’ following their release from the GSTmoiety with cyanogen bromide, was estimated to be 0.25-1 micrograms of GIP permilligram of fusion protein (based upon staining on SDS-PAGE and the publishedlimits of sensitivity of the PhastSystem electrophoreses system [Pharmacia]).Given that GIP comprised approximately 17 % of the total fusion protein byweight, one milligram of fusion protein should have yielded approximately 170 jigof GIP, assuming 100 % recovery. The vastly diminished yields suggested that thecleavage reaction was not going to completion or that the peptide was lostduring/following the cleavage process. A large molar excess of CNBr to substrate(14,000:1) did not result in significantly higher yields of cleaved product despitereports that a large molar surplus is often required to cleave a resistant methionineresidue. For example, a 3000-fold molar excess was required to cleave amethionine-serine bond in bovine pancreatic deoxyribonuclease A (Liao et al,1973).To minimize the possibility that the recombinant polypeptide was lost during thecyanogen bromide digest, all reactions, transfers and storage were carried out insiliconized glassware or polypropylene plasticware; neither porcine GIP, hGIPLeU14 nor hGlPLeu 14. Ala 23-26 interacted significantly with the reaction vessels,storage containers or transfer apparatus under these conditions. In addition, toensure that the recombinant polypeptides were not lost during the removal of theCNBr and formic acid, several different protocols were used to isolate the proteinfrom the reaction mixture. These included the evaporation of the volatile reagentsin a Speed-Vac rotaly evaporator, dilution of the reagents with water followed byrotary evaporation (Brinkmann Rotovapor Ri 10, Switzerland) or dilution of thereagents with water followed by lyophilization. None of these modificationsresulted in a significant increase in hGlPLeu 14. Ala 19-22 or hGIPLeU 14, Ala 27-30125yields yet each resulted in adequate yields of hGIPLeU j and hGIPLeU 14, Ala 23-26Hence the possibility was investigated that one or more of the fusion proteinmethionine residues was resistant to attack by cyanogen bromide.Given that GIP lies at the extreme carboxy terminus of the fusion protein, and isimmediately preceded by a methionine residue, any immunoreactive CNBrcleavage products larger than 42 amino acids (5000 daltons) must be extended atthe amino terminus beyond the methionine residue. Put another way, themethionine residue immediately preceding the GIP domain of the fusion proteinmust remain intact to generate an immunoreactive ‘product which is larger thannative GIP. The observation that all of the cyanogen bromide digests resulted inthe generation of a predominant immunoreactive 11 kDa product (Figures 25B:lane 3, 30B: lane 3, Figure 36B: lane 3, 39B: lane 2) suggested that eithermethionine number 165 or methionine 168 was being preferentially cleaved duringthe CNBr digest (Table 4). However, the possibility existed that the 11 kDafragment may have been a GIP dimer. Nonetheless, all SDS-PAGE was performedunder denaturing conditions which would discourage the formation of a multimericprotein. Therefore it appeared that the terminal methionine was significantlyresistant to CNBr attack in spite of the fact that DNA sequence analysis confirmedit had not undergone a mutation and therefore was available for cleavage.A tyrosine residue lies on the carboxy side of the terminal methionine.Apparently, certain adjacent functional groups (such as serine, threonine orcysteine) on the carboxy side of a methionine residue promote its conversion to ahomoserine residue and resultant resistance to cleavage (Schroeder et al, 1969,Doyen and Lapresle, 1979). Although there did not appear to be any publishedreports of a resistance of a methionine-tyrosine bond to CNBr attack, the scission126of this bond in the GSThGIPLeu 14 and GSLhGIPLeu 14, Ala 23-26 fusion proteinssuggested that there was no resistance to CNBr at this locus.Given that there was no evidence of an inherent immunity of the terminalmethionine to cyanogen bromide attack, it was hypothesized that an intramolecularinteraction or conformational feature conferred resistance of the GSThGIPLeu 14,Ala 19-22 or GSLhGIPLeu 14, Ala 27-30 fusion proteins to CNBr cleavage by denyingaccess of the CNBr to the terminal methionine residue. If this hypothesis wascorrect, it followed that it should be equally difficult for thrombin to access andcleave its recognition sequence which lies two amino acids upstream from theterminal methionine. Furthermore, since GSThGIPLeu 14, Ala 23-26 was readilycleaved with cyanogen bromide, it should also be readily cleaved with thrombinand therefore could act as a positive control.HPLC reverse phase profiles resulting from the cleavage of GSThGIPLeu 14, Ala 19-22’ GSThGIPLeu 14. Ala 23-26 and GSThGIPLeu 14. Ala 27-30’ with thrombin, aresummarized in Figure 42B, D and F; SDS-PAGE and western blot analysisconfirmed that the cleavage products which eluted at 10.87, 13.20 and 10.47 mm(Figure 42B, D and F respectively) were the correct predicted molecular weightfor the corresponding alanine-substituted mutant and were immunoreactive usingan anti-porcine GIP antibody. However based upon a visual comparison of theHPLC profiles, and the peak areas corresponding to each digestion product, theyield of glycine-serine-methionine (gsm)-hGIP 14, Ala 23-26 was approximately100-fold higher than that of the other two mutants (Figure 42B, D and F). Inaddition, numerous modifications to the cleavage protocol did not result insignificantly higher yields of gsm-hGIP 14, Ala 19-22 nor gsmhGIP 14. Ala 27-30(Table 5). The resistance of the fusion proteins to cyanogen bromide and127thrombin digests at adjacent but independent recognition sites supported thehypothesis that an interaction within the fusion protein andlor conformationalfeature of the fusion protein was interfering with the accessibility to the cleavagesite(s).In an attempt to generate a sufficient quantity of hGIPLeU 14, Ala 19-22 and hGIPLeU14, Ala 27-30 for biological analysis, approximately 20 mg of the associated fusionprotein was digested with thrombin. The recombinant polypeptide was purifiedover an HPLC reverse phase column; approximately 16 jig each of gsm-hGIP14, Ala 19-22 and gsm-hGIP 14, Ala 27-30 were isolated. Two milligrams of GSThGIPLCU 14, Ala 23-26 were cleaved under identical conditions, as a control, to yieldapproximately 200 jig of gsm-hGIP 14. Ala 23-26Approximately 170 jig of gsm-hGIP 14, Ala 23-26 was treated with CNBr toremove the NH2-terminus glycine/serine/methionine tripeptide. HPLC reversephase analysis indicated that 5 products were generated (Figure 43 B), one ofwhich co-eluted with uncleaved gsm-hGIP 14, Ala 23-26 (Figure 43A, C).Theoretically the digest should have resulted in three products: uncleaved gsmhGIPLeu 14, Ala 23-26’ hGIPLeu 14, Ala 23-26 and the gsm tripeptide. SDS-PAGE andwestern blot analysis indicated that each HPLC peak corresponded to animmunoreactive polypeptide with a similar molecular weight to porcine GIP(Figure 44). The generation of multiple immunoreactive products suggested thatan additional side reaction(s) was taking place.It has been reported that under acidic or neutral conditions, only two amino acidsare attacked by CNBr: methionine and cysteine (Gross, 1967). Cysteineundergoes a slow oxidation to cysteic acid in the presence of CNBr. However,128GIP does not contain any cysteine residues and therefore could not haveundergone this transformation. As suggested earlier, one consequence of CNBrexposure to methionine is the formation of methionine sulfoxide (Gross, 1967).Methionine sulfoxide is resistant to attack by CNBr and therefore the conversionof the methionine residue in recombinant polypeptide may have altered thehydrophobicity of the uncleaved material and therefore its retention time on thereverse phase column. More recently it has reported that CNBr may cleave attryptophan residues (Savige and Fontana, 1977, Huang Ct al, 1983 and Boulware etal, 1985); hGIPLeU14 Ala 23-26 contains a tryptophan residue at position number 36and therefore may be susceptible to an additional cleavage at this site.Given that the CNBr preferentially cleaves at methionine residues under acidicconditions (Gross, 1967) and that HPLC peak 2 (Figure 43B) was the predominantpeak following cyanogen bromide exposure, peak 2 was chosen for furtheranalysis. Gas phase protein sequence analysis confirmed that the first five aminoacids of the polypeptide corresponded to hGIPLeU 14, Ala 23-2& This material, likethe hGIPLeu 14, Ala 23-26 isolated earlier by a different protocol, did not potentiateinsulin release from the isolated perfused rat pancreas.The 16 tg aliquots of gsm-hGIP 14. Ala 19-22 and gsm-hGIP 14, Ala 27-30 weredigested with CNBr under identical conditions to gsm-hGIP Leul4, Ala 23-26; thedigestion products were resolved on an HPLC reverse phase column. Like thecyanogen bromide digest of hGIP Leul4, Ala 23-26’ the resultant HPLC profile of theother alanine-substituted mutants appeared to contain multiple peaks suggestingthat a side reaction(s) had taken place (Figure 46B, D). Furthermore thegeneration of multiple products, each in low yields, precluded the biologicalanalysis of the individual peaks.129V. CLEAVAGE OF THE RECOMBINANT GIP FUSION PROTEINS: ANOVERVIEWAlthough chimenc proteins are often biologically active, occasionally the isolationof native proteins is required. There are two principle methods for performing thesite-specific cleavage of a fusion protein: chemical and enzymatic proteolysis.Each has several advantages and disadvantages: the low specificity of chemicalcleavage methods limits their use since there is a reasonable probability that thetarget site exists in the recombinant product; proteases often display greaterspecificity but are more affected by steric factors which limits their accessibility totheir target sequences. Cyanogen bromide cleavage has been reported as a viablemethod for the release of a fused GIPLeU 14 product (Horn et al, 1983) yet CNBrexposure did not result in the cleavage of two of the four fusion productsgenerated for this dissertation, presumably because of steric or conformationalconstraints. Furthermore, the production of several undesirable side-productssuggests that CNBr treatment is ill-suited for any protocol designed to determinethe location of biologically-relevant amino acids. Nevertheless, the biologicalactivity of the hGIPLeU 14 mutant was not affected by the CNBr treatment. Thissuggested that the diminished biological activity, noted following the substitutionof the 23 to 26 domain of the molecule with alanme, resulted from the removal ofone or more of the side chains essential to a receptor interaction; the diminution inbiological activity was not due to a spurious alteration of the molecule by CNBr.As noted, the accessibility to thrombin was little better than that of cyanogenbromide. However, the thrombin digest of the GST-coupled fusion proteinsresulted in the generation of a homogeneous preparation of the gsm-GIP-alaninemutants as indicated by HPLC reverse phase analysis (Figures 43A and 46A, C).Hence, the successful generation and analysis of the alanine 19-22 and alanine 27-13030 substituted mutants would require two alterations to the current protocol: anincrease in the accessibility of the thrombin recognition site of the fusion proteinand the deletion of the amino acids which lie between the current thrombinrecognition site and tyrosine number 1 of GIP. This could be accomplished via arelatively simple manipulation of the present clones; the fortuitous presence oftwo restriction sites would allow for the deletion of the sequence encoding theglycine/serine/methionine fripeptide and subsequent insertion of a syntheticcassette encoding an amino terminus hydrophilic sequence and a carboxy terminusthrombin recognition sequence directly upstream to the GIP moiety of the fusionprotein (Figure 47). The inclusion of a hydrophilic spacer between the GST andGIP domains of the fusion protein should increase the accessibility to the thrombinrecognition sequences by promoting an interaction between that domain of theprotein and the surrounding media. The presence of a second thrombinrecognition sequence would also increase the probability that the hGIPLeU 14 couldbe successfully cleaved and isolated from the GST domain.The necessity for the generation of a novel expression system for two of the fourGIP mutants underscores our incomplete understanding of the mechanismsunderlying protein-protein interactions and the dynamics of protein folding. Asyet, the development of a recombinant expression system is an empirical processwhich is not necessarily amenable to predictive science. Although the expressionsystem reported in this dissertation may provide a sufficient quantity of GIP forfuture work, such as crystallographic or nuclear magnetic resonance (NMR)analysis of the molecule, perhaps the difficulties encountered here provide aninsight as to why the literature has not been inundated over the past decade withreports of recombinant protein generation.131thrombin recognitionsequenceBamFilBainHihydrophiflic sequencethrombin recognitionsequencesFigure 47: Modification strategy of the pGEX 2T-hGTP clone.4 GST ‘4 ZIPIthrombin recognitionsequencepOEX 2ThG1Pieui47—Bam IIIJKpn IKpnIsynthetic cassetteGST__hGIPIhydrophilhic sequen4 1 — /Bamifi Kpn I132VI. SYNOPSIS: THE INSULINOTROPIC DOMAIN OF GIPLimited proteolytic degradation of the GIP molecule has been used to partiallyidentify the sites contributing to the insulinotropic activity of the molecule. Theresult was a coarse map which indicated that the region of GIP responsible for itsbioactivity corresponds to residues 19 to 30 of the intact polypeptide. Based uponthe supposition that the shared biological activity of some members of theglucagon family was the result of shared sequence identity, a comparison wasmade between the residues in the 19 to 30 domain of GIP and the correspondingresidues of the other insulinotropic members of the glucagon superfamily.Truncated GLP- 1 was excluded from this analysis because displacement studiessuggest that tGLP- 1 interacts with its own receptor on the pancreatic beta cell toelicit an insulinotropic response (Graziano et al, 1993, Verchere, 1991, Usdin etal, 1993) Hence, its function as an insulin secretagogue is not the result of cross-reactivity with the GIP receptor. All of the results are summarized in Figure 48.The 19 to 30 sequence of GIP contains one of the most highly conserved regionsof the molecule. While there is no sequence identity between GIP residues 19, 28,29 or 30 and any of the other insulinotropic family members, GIP residues 20 to27 share 100 % sequence identity with those same family members (whenconsidered as a group). Individually, within this domain, GIP sequence identityranges from 75 % with glucagon to 25 % with secretin or VIP. There are noamino acids within this domain that are universally shared amongst all of thefamily members but glutamine20 and leucine26 are represented in 3 of the 4insulinotropic family members under consideration. Allowing for conservativechanges, positions 23, 26 and 27 are fully conserved amongst the respectivemembers while positions 20, 22 and 24 contain a single non-conservative change.133Residue 19 20 21 22 23 24 25 26 27 28 29 30numberGIP j val asn len len ala ginGlucagon ala.!P j val gj leu met asii thr —Secretin leu len len gly len val — — —VIP !!! JY J). !Y! i! J —RedGreenshared homology with GIPconservative substitutionsFigure 48: Amino acid sequence homologies at the carboxy terminus ofselected glucagon family members.Legend:134Taken together, these observations support a hypothesis that residues2O through27 (with the possible exception of tryptophan25 and asparagine2) are an integralcomponent of the insulinotropic domain of GIP. The observation that an alaninesubstitution of the 23-26 domain of the molecule uncouples its biological activitysupports this hypothesis. Further support is derived from the observation that asynthetic 19 to 27 fragment of secretin is an equipotent stimulant of insulinsecretion to the native full-length molecule in perifused mouse islets (Kofod et al,1991). Allowing for conservative changes, the sequence identity between theputative bioactive residues located at positions 20, 22-24, 26 and 27 of secretinand GIP is 100 %. These observations, coupled with the mutational and fragmentanalysis, provide compelling evidence that residues 20, 22-24, 26 and 27 aredirectly involved in the mediation and eventual transduction of the insulinresponse in the pancreatic beta cell.135FUTURE STUDIESAlthough experimental data has been provided which partially supports thehypothesis that GIP residues 20, 22-24, 26 and 27 mediate the insulin response atthe pancreatic beta cell, a confirmation will require the generation and analysis ofGIP analogs containing individual alanine substitutions at each of these loci. AsdIscussed earlier, the insertion of a hydrophilic linker between the GIP and GSTmoieties of the fusion construct should facilitate the cleavage of alaninesubstituted GIP analogs.Given the sequence identity between GIP and tGLP-1, it is conceivable that theproteolytic fragments generated here were stimulating an insulin response throughthe tGLP-1 receptor. However, recent work by Aldelhorst et al (1994) hassuggested that tGLP-l residues corresponding to GIP residue positions 1, 4, 6, 7and 9 are necessary for receptor binding and bioactivity while tGLP-1 residuescolTesponding to GIP residue positions 22 and 23 contribute to the structuralintegrity of tGLP-l. Clearly, the truncated G1P1730 and G1P19..30 fragments lackthe majority of the residues required for an interaction with the tGLP- 1 receptor(positions 1, 4, 6, 7 and 9). Of the remaining two residues (positions 22 and 23),only the phenylalanine at position 22 is conserved between tGLP-1 and GIP.Therefore, it is unlikely that the GIP fragments were stimulating an insulinresponse by cross-reacting with the tGLP- 1 receptor. Nonetheless, the generationof full-length GIP isoforms with individual alanine substitutions in the 19-30domain would minimize the possibility of cross-reactivity with another receptor.136In addition, this work has not addressed the possibility that multiple GIP receptorsexist on the beta cell. If so, it is possible that isoforms (or fragments) of themolecule might be generated which preferentially interact with one receptorsubtype over another. A GIP receptor has been cloned and functionally expressed(Usdin et al, 1993). If other receptor subtypes exist, their functional expression ina eukaryotic cell line would allow for the analysis of the relative contribution ofeach amino acid in GIP to the biological response. For example, the ability of thealanine-substituted GIP molecules to displace labeled GIP from each receptorsubtype could be analysed and inferences made regarding the contribution of thesubstituted amino acid to the receptor-binding process. Similarly, the use of areporter cell line, such as a cAMP reporter cell line, would facilitate the analysisof the amino acids required for the mediation of insulin release associated witheach receptor subtype.Finally, the possibility exists that the deviations in biological activity noted withhGIPLeu 14, Ala 23-26 resulted from secondary alterations to protein structure ratherthan the removal of amino acids involved in the receptor-mediated response.Although the generation of full length, alanine-substituted mutants minimizes thispossibility, three-dimensional structural analysis of the native molecule andalanine mutants would be necessary to confirm that denaturation is not responsiblefor the loss of biological activity.137APPENDIX 1LIST OF ABBREVIATIONS:ATP- adenosine triphosphateBCIP - 5 -bromo-4-chloro-3 -indolylphosphate p-toluidine saltbp - base pairsBSA - bovine serum albuminBRL - Bethesda Research Laboratories°C - degrees celciusCNBr- cyanogen bromideDNA - deoxyribonucleic aciddNTP- deoxyribonucleoside triphosphatedUMP - deoxyuracilmonophosphatedUTP - deoxyuraciltriphosphateDPP - dipeptidylpeptidaseDTT - dithiothreitolEDTA- ethylenediaminetetraacetic acidELISA - enzyme-linked immunosorbent assayFACS- fluorescence-activated cell-sorterfmol - femtomoleGI - gastrointestinalGRH - growth hormone releasing hormoneGST - glutathione S-transferasehGIP - human glucose-dependent insulinotropic polypeptide or human gastricinhibitory polypeptideIgG - immunoglobin GIR - immunoreactiveIPTG - isopropylthio-J3 -D-galactosideLB - Luria-Bertani mediumM - molarNBT - nitroblue tetrazolium chlorideOD - optical densityPAGE - polyaciylamide gelPBS - phosphate-buffered salinePEG - polyethylene glycolpmol - picomolePMSF - phenylmethyl sulfonyifluoridepsi - pounds per square inch138SDS - sodium dodecyl sulfateTAE buffer- Iris-acetate ethylenediaminetetraacetic acidTBST - Iris-buffered saline + TweenTFA - trifluoroacetic acidtGLP- 1 - truncated glucagon-like peptide 1 or glucagon-like peptide (7-37)tRNA - transfer ribonucleic acidTST- tris, saline, tweenUV - ultraviolet139REFERENCESAlderhorst K, Hedegaard BB, Knudsen LB, Kirk 0 (1994) Structure-activitystudies of glucagon-like peptide-1. 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