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A gene fusion of Cex from Cellulomonas fimi and CbhI from Trichoderma reesei Driver, Diane Patricia 1991

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A Gene Fusion of cex from Cellulomonas fimi and cbhl from Trichoderma reesei i. By Diane Patricia Driver B.Sc, The University of British Columbia, 1987 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Microbiology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September 1991 © Diane Patricia Driver, 1991 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. The University of British Columbia Vancouver, Canada Department of DE-6 (2/88) ii ABSTRACT The proteins Cex from C. fimi and Cbhl from T. reesei are exo-glucanases which are composed of separate catalytic and cellulose binding domains. When separated from one another by treatment with a protease, these domains retain their specific functions. Using polymerase chain reaction, a gene fusion was constructed which encodes a polypeptide containing the catalytic domain of Cex and the cellulose binding domain of Cbhl. During DNA sequencing of the fusion clones, an error was detected in the published Cbhl D N A sequence (Shoemaker et al. 1983b). The corrected sequence codes for two prolines, as Fagerstam (1981) determined during his earlier amino acid sequencing of Cbhl. This fusion protein, expressed in E. coli, was active on the substrates p-nitrophenyl-p-D-cellobioside, p-nitrophenyl-fi-D-lactoside, carboxymethyl cellulose and xylan, as is Cex, and was able to bind to microcrystalline cellulose but not to chitin. It can be eluted from cellulose with 8M guanidine-HC1. The fusion protein was found in the culture supernatant of E. coli cultures and presumably leaks from the periplasm in the same manner as Cex (Guo et al. 1988). Adsorption assays were conducted for the Cex/Cbhl fusion on bacterial microcrystalline cellulose (BMCC). iii TABLE OF CONTENTS Page ABSTRACT : ii TABLE OF CONTENTS iii LIST OF TABLES v i LIST OF FIGURES vii LIST OF ABBREVIATIONS viii ACKNOWLEDGEMENTS : xi 1. INTRODUCTION 1 1.1 Cellulose 1 1.2 Cellulose degradation 3 1.3 Cellulomonas fimi cellulases 5 1.4 Trichoderma reesei cellulases 8 1.5 Cellulase structural domains 10 1.6 Cellulose binding 13 1.7 Objectives of this thesis 14 2. MATERIALS A N D METHODS.. 15 2.1 Bacterial strains, plasmids, and phage 15 2.2 Media '. 15 2.3 Enzymes 15 2.4 Small scale isolation of plasmid DNA 16 2.5 Polymerase chain reaction 16 2.6 Transformation and screening 19 2.7 DNA Sequencing 19 iv TABLE OF CONTENTS continued Page 2.8 Purification of the fusion polypeptide 20 2.9 Enzyme assays 21 2.10 Electrophoretic and Western blot analysis of proteins 22 2.11 Cellulose-binding assays 23 2.11.1 Avicel 23 2.11.2 Bacterial microcrystalline cellulose 23 2.11.3 Chitin ; • 24 2.12 Chemicals and reagents 24 3. RESULTS.... 25 3.1 Construction of the gene fusion 25 3.2 Screening 30 3.3 Polypeptide characterization 34 3.3.1 Molecular weight determination 34 3.3.2 Avicel binding 34 3.3.3 Large scale preparations : 34 3.3.4 Purification from supernatant 39 3.4 Cellulose-binding assays 42 3.4.1 Determination of specific activity ^ 42 3.4.2 BMCC binding 42 3.4.3 Chitin binding 49 3.5 Activity assays 53 V TABLE OF CONTENTS continued Page 4. DISCUSSION * 55 4.1 Polymerase chain reaction 55 4.2 Protein leakage to supernatant 56 4.3 Binding assays 57 4.4 Binding and catalytic specificities 58 5. REFERENCES 59 vi LIST DF TABLES Table Page I. Primers used in PCR reactions 17 II. 10X PCR buffer 17 EI. PCR reaction mixture.' 18 IV. PCR conditions 18 V . Summary of large scale data 40 VI. Enzyme purification summary 41 VII. Binding equilibrium data 44 VIII. Binding data for Cex/Cbhl fusion on BMCC 45 IX. Summary of Kd values 51 X. Binding data for Cex/Cbhl fusion on chitin 52 XI. Activity assay results for Cex/Cbhl fusion 54 vii LIST OF FIGURES Figure Page 1. Cellulose structure 2 2. Degradation of cellulose 4 3. Domain organization of Cex and CenA 7 4. Domain organization of T. reesei cellulases 9 5. Models of CenA and Cbhl derived from SAXS analyses 12 6. PCR and cloning protocol for cex/cbhl fusion 26 7. Agarose gel of primary PCR products 27 8. Primer binding for PCR reactions 28 9. Agarose gel of secondary PCR products 29 10. DNA sequencing gel of pUC18-cbhItaii clone 31 11. Subcloning cex catalytic domain into fusion plasmid 32 12. Amino acid sequence of Cex/Cbhl fusion protein 33 13. Coomassie stained SDS PAGE of fusion showing MW 35 14. Western blot of fusion protein from JM101 and JM109 36 15. Western blot of Avicel binding 37 16. Elution of fusion protein from Avicel 38 17. Scatchard plot of BMCC binding data 47 18. Lineweaver-Burke plot of BMCC binding data 50 vm LIST OF ABBREVIATIONS A Adenine Amp Ampicillin Arg Arginine BCA Bicinchoninic acid BCTP 5-Bromo-4-chloro-3-indolyl phosphate bgl Gene coding for |3-D-glucosidase I in T. reesei BMCC Bacterial microcrystalline cellulose , C Cytosine CBD Cellulose binding domain cbhl Gene coding for 1,4-p-D-glucan cellobiohydrolase I gene in T. reesei Cbhl The cbhl gene product cbhll Gene coding for 1,4-fJ-D-glucan cellobiohydrolase II gene in T. reesei Cbhll The cbhll gene product cenA Gene coding for the endo-(3-l,4-glucanase A of C. fimi cenB Gene coding for the endo-FM/l-glucanase B of C. fimi cenC Gene coding for the endo-P-l,4-glucanase C of C. fimi CenA The cenA gene product cex Gene coding for the exo-fi-l,4-glucanase of C. fimi Cex The cex gene product CMC Carboxymethylcellulose dGTP 2-Deoxyguanosine 5'-triphosphate DMSO Dimethyl sulfoxide dNTP 2'-Deoxyribonucleoside 5'-triphosphate EDTA Ethylene diamine tetraacetate Egl The gene product of engl EgDI The gene product of englll engl Gene coding for the endoglucanase I gene of T. reesei englll Gene coding for the endoglucanase III gene of T. reesei G Guanine G A R A P Goat anti-rabbit IgG-alkaline phosphatase conjugate K a Association constant kb Kilobase Kd Dissociation constant kDa Kilodalton LB Luria-Bertani medium M U C 4-Methylumbellif eryl- P-D-cellobiopyr anoside M W Molecular weight N B T Nitro blue tetrazolium [P]o Initial protein concentration PAGE Polyacrylamide gel electrophoresis PCR Polymerase chain reaction PMSF Phenylmethyl sulphonyl fluoride pNP p-Nitrophenol pNPC p-Nitrophenyl-(3-D-cellobioside pNPL p-Nitrophenyl-P-D-lactoside Pro Proline SDS Sodium dodecyl sulphate ss Single stranded s/n Supernatant T Thymine T A E Tris-Acetate buffer TE lOmM Tris-Cl, ImM EDTA Thr Threonine UV. Ultraviolet X-gal 5-Bromo-4-chloro-3-indolyl-B-D-galactopyranoside xi ACKNOWLEDGEMENT I would like to thank Drs. R.A.J. Warren, D.G. Kilburn and R.C. Miller, Jr. for their supervision throughout this work. I am grateful to Drs. G.B. Spiegelman and J.T. Beatty for their thoughtful advice. I thank my many friends from the Department of Microbiology who have given me their friendship, laughter and advice. I especially thank my husband, Michael Abundo, for his constant support and encouragement during the past three years. I dedicate this thesis to my parents, Arnold and Eileen Driver. 1. INTRODUCTION 1.1 Cellulose Cellulose is a linear polymer consisting of up to 14,000 (3-1,4-linked glucose molecules (Coughlan 1985). About 1011 tons of cellulose are biosynthesized each year by plants, fungi, algae and bacteria. This cellulose constitutes about 50% of the bound carbon on earth (Fessendon and Fessendon 1982). This vast amount of material is constantly being degraded by fungi and bacteria. Due to this natural abundance of cellulose, it has in the past been of interest to researchers for development as a food and energy source. More recently, the potential use of bacterial and fungal cellulase enzymes in protein purification and enzyme immobilization has been given greater attention. Similar to glycogen, cellulose is a chain of glucose molecules. The two differ in that cellulose molecules are linear and have P-1,4 linkages whereas glycogen molecules are branched with cc-1,4 linkages. The glucose moieties in cellulose are in the chair configuration and each is rotated 180° along the axis of the cellulose chain (Figure 1). The disaccharide cellobiose is the basic unit of cellulose (Tonnesen and Ellefsen 1971). These long, straight chains of cellulose interact with each other through hydrogen bonding and van der Waal's forces to form a crystalline microfibril. Typically, these microfibrils are made up of about 60-70 cellulose chains, all having the same polarity (Rees et al.. 1982). Microfibrils associate to form insoluble fibres which are found closely associated with hemicelluloses and lignin in the cell walls of plants (Rees^ et al. 1982, Fan et al.. 1980). Also present in plant cell walls are proteins, lipids, pectin, starch and carbohydrates such as chitin (Ljungdahl and Eriksson 1985). The resulting material has very high tensile strength and is very difficult to ( 2 Figure 1 . Cellulose structure. From Beguin et al. 1987. (a) Stereochemical representation of a cellulose molecule. Arrows A and B represent P-l,4-linkages lying in different • planes within the cellulose fibril, (b) Organization of cellulose molecules in elementary fibrils, (c) Cross section of a wood fiber. (Adapted from Fan et al. 1980.) ( 3 degrade (Eveleigh 1987). Individual fibres are composed of both highly ordered crystalline regions and less ordered amorphous regions. Two types of cellulose considered to have very high crystallinity are Valonia cell wall and bacterial microcrystalline cellulose (BMCC) (Kulshreshtha and Dweltz 1973). If Valonia is taken to have 100% crystallinity, BMCC would have 76% and ramie cotton would have 46%. Although crystallinity is thought to be partly responsible for the resistance of cellulose to degradation, pore volume of the cellulose could be more important (Grethlein 1985, Saddler 1986) since a larger pore size would allow cellulases greater access to the inner microfibrils. 1.2 Cellulose degradation The degradation of cellulose to glucose is a complex process involving many different enzymes. Many of the genes encoding these enzymes have been cloned from bacteria and fungi (Whittle et al. 1982, Shoemaker et al. 1983a, Teeri et al. 1983, Gilkes et al. 1984a, Wolff et al. 1986, Teeri et al. 1987, Moser et al. 1989). It is not known exactly how these enzymes work together, but in the generally accepted model (Coughlan 1985), endoglucanases attack initially at an amorphous region of the cellulose fibre (Figure 2). This allows exoglucanases to act on the non-reducing ends which have been released, cleaving off a molecule of cellobiose (exocellobiohydrolases) or glucose (exoglucohydrolases). The cellobiose is then cleaved by a P-glucosidase to give glucose, which is used by the organism as a carbon and energy source. Cellobiose is known to act as an inhibitor of some cellobiohydrolases (Saddler et al. 1986), so the action of P-glucosidases can also serve to remove this end product inhibition. As this model suggests, endoglucanases and exoglucanases often act in synergy (Wood and McCrae 1978, Coughlan 1985, Eriksson and Wood 1985, Figure 2 Degradation of cellulose. From Beguin et al. 1987. Schematic representation of cellulase activity in a cellulose fibril. Individual glucose residues of the cellulose chains are represented by hexagons. The non-reducing end of a cellulose polymer is denoted by a filled hexagon. Ljungdahl and Eriksson 1985). This synergy is likely due to the endoglucanase making more sites at which the exoglucanase can act. Work with Valonia cellulose supports this model (Chanzy and Henrissat 1985). After incubation of Valonia cellulose with cellulase enzymes from T. reesei, the cellulose crystals were examined with an electron microscope. It was found that cellobiohydrolase II (Cbhll) degraded fibrils from only one end, showing that the enzyme can act only at the end of a cellulose chain as well as confirming the parallel conformation of native cellulose. In contrast, when Cbhll was combined with endoglucanase II (Eglll), there was also attack at amorphous regions, demonstrating the synergy of these two enzymes. Although the basic premise of the model appears correct, it seems to be an over-simplification. Many studies have shown that there is a greater complexity involved than this model describes (Wood et al. 1989, Enari and Niku-Paavola 1987, Hayashida and Mo 1986, Eriksson and Wood 1985, Henrissat et al. 1985, Chanzy et al. 1984, Chanzy et al. 1983). For instance, when studying synergism, Henrissat et al. (1985) showed that while Cbhll and Egll of T. reesei showed an optimal ratio for activity of 95:1, Cbhl and Egll showed a 1:1 optimum, suggesting two different types of synergy. 1.3 Cellulomonas fimi cellulases Cellulomonas fimi is a coryneform, Gram positive, facultatively anaerobic, rod shaped bacterium with an optimum growth temperature of 30°C and a genome which is 72 mole % G+C (Stackebrandt and Kandler 1979). Growth on microcrystalline cellulose (Avicel) stimulates C. fimi to produce a set of cellulolytic enzymes, whereas glycerol and cellobiose repress their production (Beguin et al. 1977, Langsford et al. 1984). The first cellulase gene to be cloned was the exoglucanase gene from C. fimi (Whittle et al. 1982) and the genes for several more cellulolytic enzymes have now been cloned from this organism. These include three (3-1,4-endoglucanases; cenA, cenB, and cenC (Gilkes et al. 1984a, Moser et al. 1989) and one (3-1,4-exoglucanase; cex (Whittle et al. 1982). C. fimi offers several advantages over other organisms such as the filamentous fungus T. reesei, even though the latter produces higher levels of cellulases. For instance, cloning of C. fimi genes is much simpler due to the fact that they contain no introns. Also, C. fimi is more resistant to end product inhibition and has much faster growth than filamentous fungi (Beguin et al. 1987). Cex and CenA are major cellulases of C. fimi. They both bind tightly to cellulose and hydrolyse it. End-products for both enzymes are predominantly cellobiose and cellotriose (Gilkes et al. 1991). CenA attacks carboxymethyl-cellulose randomly, whereas Cex degrades preferentially at terminal linkages. Comparisons can be made between Cex and CenA on the basis of their predicted amino acid seqences. Each is comprised of cellulose binding domain (CBD) of about 110 amino acid residues which is 50% conserved, and a catalytic domain of about 300 residues which does not appear to be conserved (Miller et al. 1988). The catalytic and binding domains are separated by a 20-23 amino acid "linker" region comprised of only prolyl and threonyl residues (Pro/Thr box) which is almost perfectly conserved. The linker is thought to provide flexibility and spatial separation of the two domains (Gilkes et al. 1991). Interestingly, the order of the catalytic and binding domains is reversed in these two enzymes in that the binding domain is located at the N-terminus for CenA and at the C-terminus for Cex (Figure 3). This suggests a shuffling of sequences may have occurred in the evolution of these genes (Warren et al. 1986). While both Cex and CenA are glycosylated in C. fimi, the enzymes Figure 3 Domain organization of Cex and CenA. Adapted from Beguin et al 1987. The hatched area designates the Pro/Thr box. The ordered charged region represents the catalytic domain and the irregular, low charge, hydroxyl. rich region represents the binding domain. Numbering of amino acids starts at the amino-terminus of the mature protein. ^ Pro/Thr Box CenA I r r e g u l a r , Low charge, Hydroxyl r i c h 1 Ordered, charged 112 134 418 Pro/Thr Box I r r e g u l a r , Cex Ordered, charged 1 Low charge, Hydroxyl r i c h 316 335 443 produced by E. coli from the cloned genes are not glycosylated. This lack of glycosylation does not affect catalysis or binding, but does leave the enzyme more susceptible to proteolytic attack. Proteolytic attack occurs at the C-terminal end of the Pro/Thr box. This suggests that glycosylation of the Pro/Thr box normally protects it from proteolytic cleavage (Langsford et al. 1987). 1.4 Trichoderma reesei cellulases T. reesei is a soft rot, filamentous fungus (Ljungdahl and Eriksson 1985) which produces enzymes that degrade cellulose and hemicellulose, but not lignin. T. reesei produces very large quantities of cellulolytic enzymes, accounting for more than half of all protein produced in some strains (Knowles et al. 1987). The major cellobiohydrolase, Cbhl, makes up 60% of total cellulase protein in T. reesei (Schmuck et al. 1986). Another important cellobiohydrolase, Cbhll, makes up 7%. Both cbhl and cbhll, as well as two endoglucanase genes (engl and engll) and a B-glucosidase gene (bgll) from T. reesei have been cloned and sequenced (Shoemaker et al. 1983b, Penttila et al. 1986, Teeri et al. 1987, Saloheimo et al. 1988, Barnett et al. 1991). The endoglucanases and cellobiohydrolases are separated into domains much like Cex and CenA of C. fimi (Figure 4). They consist of a large catalytic domain, a region of highly O-glycosylated amino acids (Block B) and a cellulose binding domain 35 amino acid residues long (Block A). Block B is rich in proline and hydroxyamino acids and is 50-60% identical in the T. reesei cellulases. Block A is glycine and cysteine rich and 70% identical at the amino acid level (Knowles et al. 1987). Sequence. shuffling also appears to have occurred in T. reesei cellulase genes since the binding domain is at the C-terminus of Cbhl and Egll but at the N-terminus of Cbhll and Eglll. 9 Figure 4 Domain organization of T. reesei cellulases. Adapted from Knowles et al. 1987. Block A is the binding domain. Blocks B and B' are the O-glycosylated regions. B A Cbhl Catalytic domain I A B B ' Cbhll Catalytic domain B A Egl Catalytic domain B Degradation of cellulose to glucose requires the synergistic action of all three types of enzymes; exoglucanase, endoglucanase, and B-glucosidase. In T. reesei, not only is there synergy between endoglucanases and cellobiohydro-lases, as seen in other organisms, but also between Cbhl and Cbhll (Fagerstam and Pettersson 1980, Henrissat et al. 1985). Synergy between cellobiohydrolases has also been found in Penicillium vinophilum (Wood and McCrae 1986). Cbhl and Cbhll from T. reesei can each act alone on cellulose to produce cellobiose (Fagerstam and Pettersson 1980, Chanzy et al. 1983, Chanzy and Henrissat 1985), but when working together, there is an increase in the rate of hydrolysis. This may be due to the two possible orientations of the terminal cellobiose residue (see arrows A and B of Figure I), with one enzyme being specific for one orientation and the second enzyme specific for the other (Fagerstam and Pettersson 1980). Also, Cbhl produces short fibres during hydrolysis of native cotton (Nummi et al. 1983), an action that is normally attributed to endoglucanases. Chanzy et al. (1983) also found that, like endoglucanases, Cbhl binds all over the cellulose surface rather than just at the ends as is characteristic of cellobiohydrolases. If Cbhl does indeed act as an endoglucanase, the synergy between Cbhl and Cbhll may be endo-exo rather than exo-exo synergy. Tomme et al. (1990) studied this synergy further and found evidence for a Cbhl/Cbhll complex which has a greater affinity for cellulose than each enzyme has separately. This enhanced adsorption may enable the enzymes to catalyze more efficiently, thus explaining the observed synergy. 1.5 Cellulase structural domains In both Cex and Cbhl, the separation of binding and catalytic functions into separate domains has been shown by proteolysis experiments (Gilkes et al. 1988, Langsford et al. 1987, van Tilbeurgh et al. 1986). Purified non-glycosylated Cex was subjected to limited proteolysis with a C. fimi protease, releasing two polypeptides of molecular weight 35.4 and 8 kDa. The larger polypeptide can no longer bind to cellulose but retains its ability to degrade soluble substrates such as carboxymethylcellulose and pNPC. This suggests that the large fragment contains the active site only. The smaller polypeptide seems to contain the binding domain since it retains its affinity for cellulose but is unable to degrade either crystalline cellulose or soluble substrates. Cbhl of T. reesei gave similar results after limited papain proteolysis. Both Cbhl and CenA have been analyzed by small angle X-ray scattering (Figure 5) which showed that they have a similar tadpole-shaped structure (Pilz et al. 1990, Schmuck et al. 1986, Abuja et al. 1988). The catalytic domain makes up the core while the binding domain is the long, extended tail. Despite the structural similarities between C. fimi and T. reesei cellulases, there is little v D N A sequence similarity between them. Nevertheless, the cellulose binding domains of all these proteins share the characteristic of containing few charged residues and they share a similar distribution of hydrophobic and hydroxyamino acids (Ong et al. 1989). Even though these enzymes comprise distinct catalytic and binding domains, there is some evidence that the catalytic domain is also involved in binding (Tomme et al. 1988). Furthermore, the binding domain may have a role in degradation (Greenwood et al. 1990). There is a 60% decrease in adsorption to microcrystalline cellulose by the Cbhl core as compared to the intact enzyme (Tomme et al. 1988). Much of the microcrystalline cellulose would be unavailable to the core because the Cbhl core can bind only to the most accessible regions of cellulose (Stahlberg et al. 1991). The core and the native enzyme have similar affinities for amorphous cellulose, however, 12 Figure 5 Models of CenA (A) and Cbhl (B) intact and core proteins derived from small angle X-ray scattering analysis. From Pilz et al. 1990 and Schmuck et al. 1986 B. i 1 5nm h 1 5 nm indicating an important role for the Cbhl "catalytic domain" in the binding process for certain substrates (Tomme et al. 1988). Native CenA causes fragmentation of cotton fibres into small irregular pieces, but the catalytic domain alone does not (Greenwood et al. 1990). Thus, the cellulose binding domain is required for the fragmentation of cotton fibres by CenA. Clearly, it is important to recognize the structural organization of cellulases when studying these enzymes. 1.6 Cellulose-binding Although cellulases have been extensively studied, the mechanism of binding to cellulose is, as yet, unknown. In the C. fimi enzymes, cellulose binding domains are hydroxyamino acid rich and have low charge density (Miller et al. 1988). They bind in low salt conditions and can be eluted either with water or with very high salt concentrations. This suggests that there is hydrogen bonding between cellulose binding domains and cellulose (Ong et al. 1989). Also, four regularly spaced tryptophan residues are conserved in all C. fimi CBD sequences (Gilkes et al. 1991) and may be involved in binding. Stahlberg et al. (1991) presented a theory explaining the slow adsorption rate they observed at high concentrations of cellulase. They suggested that since a molecule of cellulase is much larger than a cellobiose residue, any enzyme bound to cellulose would cover many other possible binding sites, either directly or through electrostatic repulsion. The first molecules would bind independently, but once the cellulose molecule started to get crowded, rearrangement of cellulase enzymes on the surface would be required in order for more enzyme molecules to bind. They felt that this could explain the concave Scatchard plots seen for cellulose binding. The plot would represent a gradual change from molecules binding independently at low levels to the space-limited adsorption seen as cellulase levels increased. Stahlberg et al. (1991) also noticed that at low enzyme concentrations, the intact Cbhl showed a higher association constant than either the CBD or the core and they suggested that, the intact enzyme is bound simultaneously through both of its domains. If this is true, then when one domain of the cellulase molecule binds to cellulose, the local concentration of the other domain would increase tremendously, thus promoting its binding too. They went on to suggest that, if one domain is released, it would bind rapidly at a nearby site, thus allowing movement across the cellulose surface without the enzyme being completely released from its substrate. 1.7 Objectives of this thesis Stahlberg et al. (1991) have shown that the Cbhl catalytic domain is able to bind some types of cellulose and have presented a model of Cbhl cellulose binding. The C. fimi exoglucanase, Cex, differs in that the catalytic domain is not able to bind cellulose, and the binding domain is larger than that of Cbhl. Aspects of Stahlberg's model could be studied by fusing the CBD cbhi to the Cex core, in place of C B D c e x . This thesis describes the construction of a gene fusion encoding such a polypeptide, and some of the properties of the fusion polypeptide. 2. MATERIALS A N D METHODS 2.1 Bacterial strains, plasmids and phage Plasmids pUC18-cbhI taii (provided by Tuula Teeri, Espoo, Finland) and pUC12-l.lcex (O'Neill et al. 1986), and phagemids pTZ18U (Mead et al. 1986) and pDDl (this thesis) were propagated in JM101 (Messing 1979) or JM109 (Yanisch-Perron et al. 1985). M13K07 (Vieira and Messing 1987) was used as helper phage in the production of single stranded DNA. 2.2 Media LB was made according to Maniatis et al. (1982). TYP media (Promega Catalog, Fischer Scientific, Madison, WI, USA) contained per litre, 16 g tryptone, 16 g yeast, 5 g NaCl, and 2.5 g K 2 H P O 4 . When necessary, additions to media were made as follows; ampicillin 0.1 mM, Xgal (5-bromo-4-chloro-3-indolyl-(3-D-galacto-pyranoside) 0.04 mg/mL, M U C (4-methylumbelliferyl-p-D-cellobiopyrano-side) 0.1 mM and kanamycin 0.05 mg/mL. Solid media contained 1.5% agar. 2.3 Enzymes T4 D N A ligase, T4 DNA polymerase, Taq DNA polymerase, restriction endonucleases and their buffers were purchased from either Bethesda Research Laboratories (Gaithersburg, M D , USA), New England Biolabs (Beverly, M A , USA) or Pharmacia (Uppsala, Sweden). All enzymes were used according to manufacturers' directions. D N A fragments from restriction digests or from PCR were separated on 0.7% or 1.0% agarose gels using Tris-borate-EDTA (TBE) or Tris-acetate-EDTA (TAE) buffer (Maniatis et al. 1982) and, if necessary, D N A fragments were retrieved from TAE gels using Geneclean or MerMaid kits (Bio/Can Scientific Inc., Toronto, Canada). Geneclean and MerMaid kits were used according to manufacturers instructions. 2.4 Small scale isolation of plasmid DNA Plasmid D N A was isolated from E. coli using an alkaline lysis procedure (Maniatis et al. 1982). An extra protein precipitation step was added before phenol/chloroform extraction. Ammonium acetate (10 M) was added (112.5 |iL) and precipitated proteins were removed by centrifugation. 2.5 Polymerase chain reaction Oligonucleotides (Table I) were synthesized on an Applied Biosystems Oligonucleotide Synthesizer, Model 380B by Tom Atkinson (Department of Biochemistry, UBC). They were purified according to Atkinson and Smith (1984). Primer sequences were designed to reduce mis-priming and to minimize primer-dimer production (Innis and Gelfand 1990). Considerations included primer length (usually 18-28 nucleotides), G+C content (50-60%), avoiding complementarity at the 3' ends of primer pairs, avoiding runs of C's and G's at 3' ends of primers (this can cause mispriming at G/C-rich sequences) and avoiding palindromic sequences within primers (Innis and Gelfand 1990). The composition of the PCR buffer used is given in Table II. PCR was carried out using a Cetus Thermocycler under the conditions described in Tables III and IV. Template DNA was from small scale preparations of plasmid DNA. If was digested with restriction endonucleases and the appropriate fragment was purified from a gel using Geneclean before use in the PCR reaction. Table I. Oligonucleotide primers used in PCR reactions Primer Sequence  EcoRI 1 5-GCA CTA G A A TTC C C A CGT C A C A G G GTG C A C CCG GCA-3 Hindlll 2 5'-CAT G A C A A G CTT TCG C A C GGA GCT TTA C A G GCA-3' cbhl/cex 3 5'-AGG GTT GCC GCC GCT A G G G T T / G C T CGC GCC G A A --GGC CTC CAT CAC-3 cex/cbhl 4 5'-GTG ATG G A G GCC TTC GGC GCG A G C / A A C CCT AGC--GGC GGC A A C CCT-3* Table II. 10X PCR buffer (Adapted from Innis and Gelfand 1990) Tris HC1 pH 8.3 200 mM MgCl2 5 mM KC1 250 mM Tween 20 0.5 % Gelatin 1 mg/mL 1 8 Table DX PCR reaction mixture PCR buffer IX Template D N A 2 ng primers 300 ng each MgCl2 0.5 -1.5 mM DMSO 5 -10 % Taq DNA polymerase 2 units dNTP's 50 (iM each Total Volume 100 ul Table IV. PCR conditions 30 Cycles: 96°C 15 sec. 55°C 30 sec. 72°C 1.5 min. Then 72°C 10 min. 2.6 Transformation and screening Transformants (Hanahan 1985) were screened initially on LB plates containing methylumbelliferyl-cellobiopyranoside (MUC). Cells producing Cex, Cbhl, or the fusion protein cleave off the cellobiopyranoside to release a product which fluoresces when exposed to long wave UV light. 2.7 DNA Sequencing Sequencing was done using a modified dideoxy chain termination method (Tabor and Richardson 1987). To minimize compressions, 7-deaza GTP was used instead of dGTP. DNA was produced by alkaline denaturation or using the glass fibre method as follows. Purification of ssDNA from clones in pTZ vectors was achieved by a glass fibre method (Trimbur et al., in preparation). pTZ vectors are derived from pUC vectors but contain a T7 RNA polymerase promotor and the origin of replication from fl, thus allowing the production of ssDNA when helper phage (M13K07) is added to the culture. M13K07 has a kanamycin resistance gene inserted such that its own ssDNA production is slowed. Also, it has inserted in its genome a plasmid origin of replication so that the double stranded form is in high enough concentration in the cell to produce phage proteins for replication and packaging of the pTZ vector. JMlOl-pDDl was grown with M13K07 overnight in TYP medium containing kanamycin. Then 1.5 mL of the culture was centrufuged to remove cells, and phage particles were precipitated from the culture supernatant by the addition of 0.5 M ammonium acetate, 3.3% PEG. After a 30 minute incubation, phage particles were pelleted and resuspended in 20 JLLL TE. Sodium perchlorate (4 M) was added (200 u.L) and the sample was filtered through a Whatman G F / C glass fibre filter (Maidstone, England) which binds the DNA. Salts were removed by washing the filter with 70% ethanol, and then the filter was dried and DNA was eluted with 20 uL of 0.1X TE. When sequencing pUC18-cbhItaii, mini-prep DNA was used to produce denatured DNA suitable for sequencing (Kraft et al. 1988). The first part of this procedure was the same as the small scale isolation of plasmid D N A protocol (See Section 2.4) except that before addition of ammonium acetate, DNase free RNase was added to a final concentration of 50 | ig/mL and the solution was incubated at 37°C for 30 minutes. After dessication of the ethanol precipitated DNA, the pellet was re-dissolved in 16.8 uL of distilled water and DNA was again precipitated by the addition of 3.2 uL of 5 M NaCl and 20 uL of 13% PEG and incubation on ice for 30 minutes. After centrifugation, the pellet was washed with 1 mL of 70% ethanol and dessicated, then re-dissolved in 20 uL distilled deionized water. DNA was denatured in by a 10 minute incubation with 2 (iL of 2 M NaOH, 2 mM EDTA. DNA was then ethanol precipitated, washed with 70% ethanol and dessicated. When ready to sequence, this pellet was re-dissolved in 10 [iL sequenase buffer containing the appropriate primer and sequencing was carried out as usual. Sequencing primers were either the universal primer (Pharmacia, Uppsala, Sweden) or PCR primer #2 (Table I). 2.8 Purification of the fusion polypeptide A I L culture of JMlOl-pDDl was grown for 16-22 hours at 37°C. Cells were removed by centrifugation at 8000 rpm for 30 minutes. Then 15 g of Avicel microcrystalline cellulose PH-101 (FMC Int., County Cork, Ireland) was added per litre of culture supernatant liquid and the suspension was stirred slowly overnight at 4°C. The Avicel was allowed to settle and most of the culture supernatant liquid was poured off, and the Avicel was recovered by filtration through a Whatman G F / C glass fibre filter. The Avicel was washed repeatedly by resuspension in 1 M NaCl until the A280nm of the supernatant liquid was less than 0.1 (about 6 washes). After each wash, Avicel was recovered by filtration through a glass fibre filter. Then Avicel was washed two times with 50 mM potassium-phosphate buffer (pH 7) and the cellulase was eluted with 8 M guanidine-HCl (40 mL per 15 g Avicel). Eluted enzyme was concentrated and desalted using Amicon apparatus (Amicon Division, W.R. Grace and Co., Beverly, M A , USA). Gary Lesnicki (U.B.C. Biotechnology Dept) prepared large scale cultures in LB with ampicillin using a 30 L fermenter (Chemap A G Switzerland). 2.9 Enzyme assays Activity of the fusion protein was determined by measuring release of p-nitrophenol (pNP) from p-nitrophenyl-B-D-cellobioside (pNPC) during incubation with the enzyme (Gilkes et al. 1984b). To determine activity inside cells, E. coli were either toluenized or sonicated, followed by an assay for pNPCase activity. Toluenization involved washing the cells two times with 50 mM potassium-phosphate buffer (pH 7), then resuspending in 0.3 mL buffer, adding 5 U.L of 1:9 toluene:ethanol and mixing for one minute. The cell suspension was then used directly in the pNPCase assay. Cells were sonicated for three 30 second bursts separated by 1 minute cooling on ice. The Sonifier Cell Disrupter 350 (Branson Sonic Power Co.) was used on setting 3, continuous, using a tapered micro tip probe. After sonication, PMSF was added to a concentration of 0.2 mg/mL. Cell debris was removed by centrifugation and the supernatant fluid assayed for pNPCase activity. The pNPCase assay was done by adding 0.5 mL of diluted enzyme (in 50 mM potassium-phosphate p H 7 buffer) to 0.5 mL of 12.5 mM pNPC. Both were prewarmed before being combined together and were incubated for approximately 30 minutes at 37°C. The enzyme cleaves off the cellobioside leaving the yellow coloured pNP. The reaction was stopped and the yellow colour intensified by the addition of 0.5 mL 1 M Na2C03. If cells were present, they were removed by centrifugation before absorbance was read at 410 nm. Comparison to a standard curve of pNP allowed determination of the nmoles pNP/mL in the reaction mixture and the activity was expressed in units/mL where one unit is the amount of enzyme required to release 1 pmole pNP from pNPC in 1 minute at 37°C at p H 7. Protein concentration was measured using the bicinchoninic acid (BCA) assay (Pierce Chemical Company, Rockford, Illinois, USA) or the Bio-Rad dye binding assay (Bio-Rad Laboratories, Richmond, CA). Specific activity (U/mg) was determined by dividing activity (U/mL) by protein concentration (mg/mL). This value could be used to determine protein concentration of a sample by doing an activity assay on the sample and then converting to protein concentration by dividing by specific activity ([U/mL] / [U/mg] = mg/mL). 2.10 Electrophoretic and western blot analysis of proteins A Bio-Rad Mini-Protein gel apparatus was used to run SDS poly-acrylamide gels for separation of proteins. The stacking gels were 4% acrylamide p H 6.8, and the separating gels were 12% acrylamide pH 8.8. Gels either were stained with Coomassie blue or were used for western blotting. For western blots, after blotting to nitrocellulose and blocking residual sites with BSA, proteins were incubated with a 1/2000 dilution of polyclonal rabbit anti-Cex antibody (preabsorbed to cell extract from JM101-pTZ18U). Then they were washed and incubated with a 1/7000 dilution of goat anti-rabbit IgG coupled to alkaline phosphatase (GARAP). The blot was washed again and was then developed by incubation with 50 |!g/mL BCIP and 100 | ig/mL NBT (pH9.6). Proteins to be used on mini-gels were either purified as in Section 2.8 or were prepared as the SDS boiling method by removing cells by centrifugation, resuspending the pellet in IX SDS loading buffer and boiling. Then membranes were removed by centrifugation and the supernatant liquid was loaded onto the gel. 2.11 Cellulose-binding assays 2.11.1 Avicel Avicel binding assays involved binding proteins to Avicel in 50 mM potassium phosphate buffer at 4°C for 1 hour, then spinning out the Avicel. The supernatant was run on a gel to determine the unbound proteins and the Avicel was boiled in IX SDS loading buffer to release bound proteins. After removing Avicel by centrifugation, these proteins were run on a gel. 2.11.2 Bacterial microcrystalline cellulose (BMCC) BMCC was prepared by Emily Kwan (UBC Microbiology Department) from a 14 day culture of Acetobacter xylinum. The cellulose pellicle was cut into small pieces and bleached with NaOH. It was then hydrolyzed with HC1 and disrupted in a blender. This was repeated three times, then the cellulose was washed extensively with water. It was resuspended in 50 mM potassium phosphate buffer p H 7 to a concentration of 2 mg/mL. The concentration was determined by removing a known volume of the suspension, drying it and measuring the dry weight. Binding assays were done in triplicate as follows. Enzyme concentrations varied from 0.1 to 22 (ig/mL in a final volume of 1.5 mL in 2 mL microcentrifuge tubes. Binding experiments were carried out at 4 °C . Enzyme was combined with 1 mg/mL BMCC for one minute, then the cellulose was removed by centrifugation and the supernatant assayed for pNPCase activity. 2.11.3 Chitin Chitin from crab shells (Sigma, St. Louis, MO) was prepared by Emily Kwan (UBC Microbiology Department) by washing repeatedly in 50 mM potassium phosphate pH 7 buffer. Binding assays were done in duplicate using enzyme concentrations varying from 9.3 to 279.5 L i g / m L in a final volume of 0.75 mL. Binding was done using 1 mg/mL chitin for 100 minutes at 4°C with mixing. Chitin was then removed by centrifugation and the supernatant liquid assayed for pNPCase activity. 2.12 Chemicals and reagents Chemicals and reagents were purchased from the following chemical companies: BDH (Toronto, Ont), Sigma Chemical Co. (St. Louis, MO), Difco (Detroit, MI), Boeringer-Mannheim Canada Ltd. (Laval, Quebec), BBL Microbiology Systems (Cockeysville, MD), J.T. Baker Chemical Co. ' (Phillipsburg, NJ), and Bio-Rad Laboratories (Richmond, CA). 3. RESULTS 3.1 Construction of the cex/cbhl gene fusion Fusion of the cex sequence encoding the catalytic domain with the cbhl sequence encoding the binding domain was achieved through polymerase chain reaction (Figure 6). Primary PCR involved amplification of the cex catalytic domain and the cbhl binding domain separately (Figure 7) using primers 1 and 3 for cex and primers 2 and 4 for cbhl (Figure 8). A restriction site (EcoRl) was added at the 5' end of the cex fragment and, at the 3' end, a sequence was added which is homologous to 21 bases at the 5' end of the cbhl PCR product. Similarly, a Hindlll site was added to the 3' end of the cbhl binding domain during PCR and at the 5' end, a sequence was added which is homologous to 24 bases at the 3' end of the cex PCR product. Thus, the products of primary PCR could be combined in a second PCR with the homologous regions acting as primers to allow fusion of the two fragments. Primers 1 and 2 were included in a secondary PCR reaction to allow amplification of the fusion product (Figure 9). Before being used in the secondary PCR reaction, primary PCR products were purified using Geneclean or MerMaid in order to remove primers used in primary PCR, and other unwanted products from primary PCR. After secondary PCR, the product was again purified using Geneclean in order to remove DMSO, PCR buffer and dNTP's which could interfere with the restriction endonuclease digestion or the ligation. The fusion fragment was digested with EcoRl and Hindlll, ligated into pTZ18U which had been digested with the same enzymes, and transformed into JM101. Figure 6 PCR and cloning protocol for cex/cbhl gene fusion. EcoRl Apal Ampr EcoRIilSmal PvuII cbhICBD cbhl catalytic fragment BamHl Hindlll PvuII cex catalytic domain Xmal Apal Apal digest Geneclean 1.5 kb fragment Apal Amp"" PvuII digest Geneclean 0.9 kb fragment 1.5 kb I 0.9 kb cex catalytic cex CBD Primary PCR EcoRl cbhl cbhl catalytic CBD fragment Primary PCR 1.1 kb cex catalytic j 21 bp cbhl CBD • 0.2 kb Secondary PCR :_! 24 bp cex cbhl catalytic CBD domain Hindin ' EcoRl 13 kb HindlO cex catalytic domain cbhl CBD EcoRl and Hindlll digests pTZ18U Xmal 27 Figure 7 Agarose gel of primary PCR products. lOpL of each PCR reaction is loaded onto the gel. Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 Lane 7 0.2 kb Cbhl fragment 0% DMSO, 2.5 mM M g C l 2 0.2 kb cbhl fragment 5% DMSO, 2.5 mM MgCl 2 0.2 kb cbhl fragment 10% DMSO, 2.5 mM MgCl 2 0.7 p.g of Ddel digest of pUC12 DNA. Fragment sizes in # of base pairs: 166,235,409,426,540, and 910. 0.8 p.g of Pvull digest of X DNA. Fragment sizes in # of base pairs: 63,141,209,343,468,532,579,636,1701,2296, 3638, 3916,4198, 4268,4421, and 21090. 1.1 kb cex fragment 10% DMSO, 1.5 mM M g C l 2 1.1 kb cex fragment 10% DMSO, 2.0 mM M g C l 2 1 2 3 4 5 6 7 Figure 8 Location of primer binding for PCR reactions. Numbers refer to the numbers given to primers in Table I. Primary PCR cex catalytic cex CBD !wlsi cbhl cbhl catalytic CBD Secondary PCR EcoRI FT cex catalytic HindHI cbhl CBD 29 Figure 9 Agarose gel of secondary PCR products IOJIL of each PCR reaction is loaded onto the gel. Lane 1 1.3 kb fusion, 0% DMSO, 0.5 mM M g C l 2 Lane 2 1.3 kb fusion, 0% DMSO, 1.5 mM MgCl 2 Lane 3 1.3 kb fusion, 0% DMSO, 2.5 mM MgCl 2 Lane 4 1.3 kb fusion, 5% DMSO, 0.5 mM MgCl 2 Lane 5 1.3 kb fusion, 5% DMSO, 1.5 mM MgCl 2 Lane 6 0.3 |ig of X DNA digested with Hindlll. Fragment sizes in # of base pairs: 564,2027,2322,4361,6557, 9416,23130. Lane 7 0.4 ug of X DNA digested with Hindlll and EcoRl. Fragment sizes in # of base pairs: 564, 831, 983,1330,1584, 1904,2027,3530,4277,4973,5148, and 21226. Lane 8 0.7 u.g of Ddel digest of pUC12 DNA. Fragment sizes in # of base pairs: 166, 235,409,426,540, and 910. Lane 9 1.3 kb fusion, 5% DMSO, 2.5 mM M g C l 2 Lane 10 1.3 kb fusion, 10% DMSO, 0.5 mM M g C l 2 Lane 11 1.3 kb fusion, 10% DMSO, 1.5 mM M g C l 2 Lane 12 1.3 kb fusion, 10% DMSO, 2.5 mM M g C l 2 1 2 3 4 5 6 7 8 9 101112 AMM M M 4Hlr 3.2 Screening Initial screening of transformants was done on M U C / L B / A m p plates. Colonies which fluoresced under UV light were grown up and their plasmids sequenced. A number of mutations, mostly G to A transitions, were discovered when comparing the sequences with the published D N A sequence of cbhl (Shoemaker et al. 1983b). There was approximately 1 mutation every 80 base pairs sequenced. An error was also detected in the published D N A sequence of Cbhl (Shoemaker et al. 1983b). I found the sequence C C G CCT which codes for two prolines. The published DNA sequence for the same region (nucleotides 1714-1716) is CGT, which codes for arginine. Re-sequencing the original pUC18-cbhl t a ii clone also revealed C C G CCT, not CGT (Figure 10). When Shoemaker et al. (1983b) had sequenced the DNA, they noticed that there was a discrepancy between their sequence (which coded for Arg) and the amino acid sequence (ProPro) which had been determined by Fagerstam (1981), but they assumed the DNA sequencing results to be correct. Only the C-terminus of my fusion gene was sequenced, including the whole cbhl binding domain and the junction between cbhl and cex. As stated earlier, PCR introduces many mutations during synthesis. Rather than sequence the whole fusion, I removed, using the restriction enzymes Xmal and Styl, the section of the cex catalytic domain which had not yet been sequenced and I replaced it with the same fragment from the original pUC 12-cex clone (Figure 11). The final cex/cbhl fusion clone (pDDl) had one mutation, but it was a silent mutation from GGC to AGC, both of which code for alanine. The amino acid sequence of the Cex/Cbhl fusion is given in Figure 12. Figure 10 DNA sequencing gel of pUC18-cbhItaii clone. Sequences shown are the non-coding strand. Comparison with sequence in the literature is shown (Shoemaker et al. 1983b). A C G T Arg Published sequence: GGGTGGTGGTGGTGCC / A C G /GTTT Pro Pro Actual sequence: GGGTGGTGGTGGTGCC/AGGCGG/GTTT 32 Figure 11 Procedure for subcloning non-PCR cex catalytic domain into fusion plasmid. Apal Hindlll Xmal and Styl digests Geneclean fragment Xmal and Styl digest Geneclean Xmal Hindlf l Xmal 3 3 Figure 12 Amino acid sequence of Cex/Cbhl fusion protein. Leader sequence is not included in this figure. Junction between the catalytic domain and Block B is denoted by a slash. 5 10 15 20 25 30 1 A T T L K E . A A D G A G R D F G F A L D P N R L S E A Q Y K 31 A I A D S E F N L V V A E N A M K W D A T E P S Q N S F •s F 61 G A G D R V A S Y A A D T G K E L Y G H T L V w H S Q L P D 91 W A K N L N. G S A F E S A M V N H V T K V A D H F E G K • V A 121 S W D V V N E A F A D G D G P P Q D S A F Q Q K L G N G Y I 151 E T A F R A A R A A D P T A K L C I N D Y N V E G I N A K S 181 N S L Y D L V K D F K A R G V P L D C V G F Q S H L I V G Q 211 V . P G D F' R Q N L Q R F A D L G V D V R I T E L D I R M R T 241 P s D A T K L A T Q A A D Y' K K V V Q A G M Q V T R C Q G V 271 T V W G I T D K Y S W V P D V F P G E G A A L V W D A S Y A-301 K K P A Y A A V M E A F G A S / N P S G G N P P G G N P P G T 331 T T T R R P A T T T G S S P G P T Q S H Y G Q C G G I G Y S 361 G P T V. C A S G T T C Q V L N P Y Y S Q C L 3.3 Polypeptide characterization 3.3.1 Molecular weight determination To determine protein size, the protein was run on SDS-PAGE and a western blot was done. The protein had an apparent molecular weight of approximately 41 kDa's (Figure 13) as predicted in calculations done from the amino acid sequence. . Both JM101 and JM109 strains had been transformed with the plasmid p D D l . On a western blot (Figure 14), JM109 showed fewer degradation products, so this strain was used in most of the following experiments. 3.3.2 Avicel binding Since it was hoped to purify the polypeptide by affinity chromatography on Avicel, I performed preliminary studies to determine if the fusion protein would bind well to Avicel. The 41 kDa fragment bound to Avicel, while a degradation product of about 30 kDa (presumably the Cex catalytic domain) did not bind (Figure 15). Cex can be eluted from Avicel with. 8 M guanidine-HCl, but it was not clear from the literature how to elute the Cbhl binding domain from cellulose. Purification of Cbhl is usually done by affinity chromatography using a ligand coupled to Sepharose (van Tilbeurgh et al. 1984). I tested elution of the fusion from Avicel using water, 6 M urea and 8 M guanidine-HCl (Figure 16). Only guanidine-HCl was found to give satisfactory elution. 3.3.3 Large scale preparations Soon after beginning large scale preparations, it was discovered that the fusion protein was present in the culture supernatant. Examination of this protein showed it to have the same apparent molecular weight, pNPCase activity and Avicel binding ability as the fusion product harvested from within 3 5 Figure 13 Coomassie stained SDS PAGE of fusion protein showing molecular weight Lane 1 Molecular weight standards Lane 2 Purified Cex/Cbhl fusion protein. 1 2 205k. ID SL 36 Figure 14 Western blot of Cex/Cbhl fusion protein from JM101 and JM109. Proteins are prepared by the SDS boiling method; the cell pellet from 2 mL of a culture is boiled in 200 |jL of IX SDS loading buffer. 10 uL of this preparation is loaded into each well. The fusion protein is approximately 41 kDa, Cex is approximately 46.5kDa. Lanes 1 and 2 Cell extract from JM109-pDDl Lane 3 Cell extract from JM101-pTZ18U Lane 4 Cell extract from JMlOl-pDDl Lane 5 Cell extract from JM83-pUC13 Lane 6 Cell extract from JM83-pUC12-l.lcex Lane 7 Molecular weight standards: 15,18, 28.5,44, 71, 111, and 206 kDa. 37 Figure 15 Western blot of Avicel binding experiment for Cex/Cbhl fusion protein. Cell extract was prepared by sonication. Cells from 3mL culture were pelleted by centrifugation and were resuspended in 600 u.L buffer. After sonication as in Materials and Methods, 100 (iL was added to Avicel and this was incubated on ice for 1 hour. Avicel was removed by centrifugation leaving the unbound proteins in the liquid supernatant. To remove protein from Avicel, 100 pL of IX SDS loading buffer was added to the Avicel and was boiled for 1 minute. Lane 1 Molecular weight standards: 15,18,28.5,44, 71, 111, and 206 kDa. Lane 2 8 pL of cell extract Lane 3 8 uL of unbound protein Lane 4 8 pL of protein removed from Avicel 38 Figure 16 Elution of Cex/Cbhl fusion protein from Avicel using water (A), 6M urea (B), or 8M guanidine-HCl (C). 1.5 mL of culture supernatant liquid was incubated with Avicel. Avicel was removed by centrifugation and washed repeatedly, then protein was eluted as indicated. Avicel is resuspended in 50 (iL IX SDS loading buffer and boiled. This was loaded onto the gel to give 'Avicel bound' fraction. Arrowheads indicate the fusion protein. A. Water Elution Lane 1 Molecular weight standards: 15, 18, 28.5,44,71, 111, and 206 kDa. Lane 2 5 |iL Culture s/n Lane 3 10 |iL Avicel bound Lane 4 10 |iL Avicel bound after high salt wash Lane 5 10 uL Avicel bound after low salt wash Lane 6 10 ^L Avicel bound after water washes B. Urea elution Lane 1 Molecular weight standards: 15, 18, 28.5,44, 71, 111, and 206 kDa. Lane 2 5 p.L Culture s/n Lane 3 5 (J.L Avicel s/n (unbound) Lane 4 10 (iL Avicel bound Lane 5 10 |J.L Avicel bound after 6M urea washes C. Guanidine-HCl elution Lane 1 10 (iL Avicel bound Lane 2 10 |iL Guanidine washed Avicel the cells. Thus, subsequent purification was done from the supernatant fluid. Large scale protein preparations from 10-60 litre cultures were attempted but with little success. Although cell growth was adequate, fusion protein yield was low in all cases. Many changes were made to growth conditions such as varying airflow (0-15 L/min.), using p H control, changing the media source (BBL or Difco), and not including antifoam in the culture medium. None of these changes improved enzyme, yield. Also, we ran parallel experiments in the fermenter and in 2 litre flasks using the same media and inoculum but, without exception, the flask cultures produced more fusion protein than the fermenter cultures. Table V summarizes yields from various large scale preparations. The results can be compared to an average of 20-50 units per litre in the supernatant of 1 litre cultures. 3.3.4 Purification from supernatant Cultures were sampled at intervals to determine the best time for harvesting, but the results were variable, even when identical runs were done at the same time using the same inoculum and conditions. Generally, an overnight culture of 16-22 hours gave the highest activity, after which time, activity dropped off gradually. Although activity both in the supernatant fluids and within the cells was low in large scale preparations, 1 litre cultures produced protein well with about one half of the total fusion protein being found in the supernatant fluid. Generally, each 1 litre culture produced about 20-50 units of pNPCase activity. Much of this was lost during purification, usually leaving about a 20% yield in the final product after a 35 fold purification (Table VI). However, as much as 40% of the activity was found in the Avicel supernatant, mostly in the form of degradation products. Thus, the recovery of intact polypeptide was about 35%. Also, a precipitate was formed Table V. Summary of data from large scale cultures. Those samples not tested are marked n/d (not done). Run Size (litres) Time (hours) ODeoo U / L (cells) U / L (s/n) U / L (total) 1 10 6.5 3.0 7.0 n/d n/d 2 60 9.0 6.0 0.2 n/d n/d 3 20 6.5 8.5 4.5 5.0 9.5 4 20 22 4.4 0.2 0.3 0.5 5 20 30 3.7 0.9 0.4 1.3 6 20 25 5.9 2.5 4.0 6.5 7 20 18 4.3 n/d 0.5 n/d 8 20 22 2.1 n/d 7.4 n/d 9 20 19 2.0 n/d 6.0 n/d 10 20 36 2.3 10 5.0 , 15 Table VI. Example of an enzyme purification summary for Cex/Cbhl fusion protein. Purified from a 4.4 L culture. Sample 1: s/n from culture after 30 min spin Sample 2: s/n after binding to Avicel' Sample 3: s/n from first wash of Avicel Sample 4: sample after elution from Avicel with 8 M guanidine-HCl, then desalting and concentration using Amicon apparatus Sample Volume (mL) units/ mL total U protein (mg/mL) total protein (mg) Specific activity (U/mg) Yield (%) Fold purifi-cation 1 4400 0.0226 99.5 0.125 563 0.177 100 0 2 4400 0.0090 39.6 0.128 563 0.07 40 -3 250 0.0037 0.925 0.037 9.25 0.10 0.93 -4 0.99 19.8 19.6 3.179 3.15 6.22 19.7 35 during the dilution step of the Amicon procedure. This precipitate could be partially redissolved in 2 mM N H 4 O H and some activity was recovered. Even so, much of the total activity remained unaccounted for and could still have been in the insoluble precipitate. 3.4 Binding Assays 3.4.1 Determination of specific activity Initially, I attempted to determine protein concentration using the A205/A28O method (Scopes 1974), but the results were irreproducible, and were not in' agreement with the predicted results calculated from the protein's amino acid content. I then determined protein concentration using the Bio-Rad dye binding assay and the BCA assay, which gave similar results. The value determined using the BGA assay was used in the calculation of specific activity. Specific activity was determined to be 6.2 units/mg. In comparison, Cex specific activity on pNPC was about 14 units/mg. During the binding assays, protein concentration was determined by first measuring pNPCase activity, then using the specific activity to calculate the protein concentration: [P] (mg/mL) = Activity (U/mL) / Specific activity (U/mg). 3.4.2 BMCC binding It is important to ensure the stability of the enzyme during binding and during the activity assay afterwards. I found that at the low concentrations which I was using, the addition of bovine serum albumin (O.lmg/mL) served to stabilize the enzyme. Also, the enzyme lost activity rapidly at 30°C or room temperature, so binding experiments were done at 4°C. Since pNPCase assays required a temperature of 37°C, the length of time for the assay was kept to a minimum. I found that after 10 minutes at 37°C, activity began to decline, so 10 minute continuous assays were done using the Hitachi U-2000 spectro-photometer which takes continuous readings and plots these readings on a graph. It is also important in binding assays to ensure that the enzyme achieves maximal binding. Table VII shows that at the highest concentration used in my binding assays, maximum binding on BMCC at 4°C was reached in less than,one minute. Activity measured in the supernatant liquid after binding represents 'free' or unbound enzyme expressed in U / m L which I then converted to mmole/mL using specific activity and molecular weight values. Since the original protein concentration ([P]o) was known, concentration of 'bound' enzyme could easily be determined by subtracting 'free' enzyme from [P]o. Data are shown in Table VIII. Figure 17 shows these data graphed as a Scatchard plot. As seen for both Cbhl (Woodward et al. 1988) and Cex (Greenwood et al. 1990), the plot for the fusion protein is concave. Another way to treat these data is to express them as a Lineweaver-Burke plot. This plot emphasizes the lower concentration range of the adsorption data. The Lineweaver-Burke equation is derived from the Langmuir isotherm as follows. The equilibruim reaction of cellulose and cellulase is given by Ka Free Lattice Sites (FLS) + Free Ligand(FL) Bound Ligand(BL) 1. From the law of mass action we can write K a= rBLl [FLS][FL] 2. The concentration of free lattice sites can be written as [FLS]=[FLS]initial-[BL] 3. Table VII. Determination of maximum binding for Cex/Cbhl fusion on BMCC. Determination of maximum binding using 0.022 mg/mL enzyme combined with 1 mg/mL BMCC, agitated at 4°C for the time given. Time is in minutes allowed for binding before the BMCC was removed by centrifugation and s/n was assayed for pNPCase activity, units given are U / m L pNPCase free in the s/n. Two experiments are shown; one with a maximum time of 280 minutes, and one of 1000 minutes. Sample 1 min. 15 min. 100 min. 280 min. no BMCC 0.130 0.133 0.135 0.129 with BMCC 0.023 0.023 0.021 0.023 Sample 1 min. 15 min. 1000 min. no BMCC 0.128 0.120 0.128 with BMCC 0.022 0.023 0.022 Table VIII. Adsorption data for Cex/Cbhl fusion on BMCC. Sample number \P]o (mM) Free (mM) Bound (mmole/ g BMCC) Bound/Free (mL/ mg BMCC) la 2.610e-6 1.659e-7 2.444e-6 14.735 lb . 2.610e-6 1.659e-7 2.444e-6 14.735. lc 2.610e-6 1.759e-7 2.434e-6 13.834 2a 6.525e-6 5.629e-7 5.962e-6 10.590 2b 6.525e-6 5.328e-7 5.992e-6 11.246 2c 6.525e-6 A.777e-7 6.047e-6 12.660 3a 1.305e-5 1.173e-6 1.188e-5 10.127 3b 1.305e-5 1.005e-6 1.204e-5 11.981 3c 1.305e-5 1.117e-6 1.193e-5 10.684. 4a 1.957e-5 2.122e-6 1.745e-5 8.224 4b 1.957e-5 1.675e-6 1.790e-5 10.683 4c 1.957e-5 2.01 le-6 1.756e-5 8.736 5a 2.610e-5 2.346e-6 2.375e-5 10.127 5b 2.610e-5 2.569e-6 2.353e-5 9.158 5c 2.61 Oe-5 2.681e-6 2.342e-5. ; 8.736 6a 5.220e-5 5.808e-6 4.639e-5 7.987 6b 5.220e-5 5.918e-6 4.628e-5 7.821 6c 5.220e-5 6.030e-6 4.617e-5 7.657 .continued on following page Table VIII continued... Sample number [P]o (mM) Free (mM) Bound (mmole/ g BMCC) Bound/Free (mL/ mg BMCC) 7a 1.044e-4 1.396e-5 9.044e-5 6.477 7b v 1.044e-4 1.005e-5 9.435e-5 9.385 7c 1.044e-4 1.415e-5 9.025e-5 6.379 8a 1.568e-4 2.094e-5 1.359e-4 6.487 8b 1.568e-4 2.122e-5 1.356e-4 6.388 8c . 1.568e-4 2.122e-5 1.356e-4 6.388 9a 2.089e-4 2.904e-5 1.799e-4 6.193 9b 2.089e-4 2.644e-5 1.825e-4 6.902 9c 2.089e-4 3.090e-5 1.780e-4 5.760 10a 3.262e-4 3.909e-5 2.871e-4 7.344 10b 3.262e-4 3.965e-5 2.865e-4 7.227 10c 3.262e-4 4.580e-5 2.804e-4 6.123 11a 4.436e-4 6.534e-5 3.783e-4 5.789 l ib 4.436e-4 6.758e-5 3.760e-4 5.564 11c 4.436e-4 1.005e-4 3.431e-4 3.413 12a 5.477e-4 1.117e-4 4.360e-4 3.903 12b 5.477e-4 9.532e-5 4.524e-4 4.746 12c 5.477e-4 1.296e-4 4.181e-4 3.227 Figure 17 Adsorption data of Cex/Cbhl fusion on BMCC; graphed as a Scatchard plot. 'A' shows all data. 'B' shows only the four lowest concentrations. The is calculated by taking the negative inverse of the slope. A. o o S m E a> u. c • o to B Bound/Free (mlVmg BMCC) 100 200 300 400 Bound (nmole/g BMCC) 5 0 0 B. •o c o m 16 14 O O s m E S 12 -« 10 -y= 14.380-0.30085X RA2 = 0.723 • • • • • Bound/Free (mL/mg BMCC) 1 0 Bound (nmole/g BMCC) 20 48 This can be substituted into equation 2 to give the Langmuir isotherm rBLl = fFLSli K a [FL1 (1+KafFL]) 4. Taking the inverse of this equation gives the Lineweaver-Burke equation 1' = 1 + 1 [BL] [FLS]iKa [FL] [FLSJi 5. The binding data can be graphed as a Lineweaver-Burke plot with 1/[FL] on the X axis and 1/[BL] on the Y axis. From the Lineweaver-Burke equation, we can see that the slope of this line would be l / (Ka [FLS]i) and the Y intercept would bel/[FLS]i. The Langmuir isotherm, which is the basis of both the Scatchard and Lineweaver-Burke plots, assumes that one ligand interacts with only one binding site with no cooperativity. We know, however, that a cellulase molecule is much larger .than a glucose or cellobiose residue of a cellulose chain. Thus, each cellulase molecule blocks more than one potential binding site and the assumption that each binding site can be bound is not valid. This problem was addressed by Erik Jervis (UBC Biotechnology Dept.). He proposed the following method of dealing with the problem. Assume that one ligand molecule excludes the binding of more than one potential binding site by other ligand molecules. The following equation estimates the probable number of free lattice sites [FLS]=P(B) [FLS]i 6. where P(B) is the probability of finding a free site and [FLS]i is the initial concentration of free lattice sites. Substituting equation 6 into equation 2 gives the equation ' [BL]=P(B)[FLS]iKa[FL] 7. This can be rearranged into the Lineweaver-Burke equation 1 1  [BL] P(B) [FLSJi K a [FL] 8. The slope would then be , • 1 . P(B)[FLS]iKa 9. At very low bound ligand concentrations, P(B) would approach one, thus giving a slope of 1 '. ,• or K H [FLS]iKa 1 [FLSJi 10. Thus, it is not possible to determine the absolute adsorption affinity. However, this value can be used for the determination of relative association constants for a given preparation of cellulose such that the affinities of various cellulases can be compared. Figure 18 shows the BMCC binding data expressed as a Lineweaver-Burke plot. The slope of this plot was used to determine a relative dissociation constant (Kd/[FLS]i). Table IX summarizes these relative dissociation constants calculated for Cex and for the Cex/Cbhl fusion, as well as dissociation constant values from the literature for Cbhl and CBDCbhi (Stahlberg et al. 1991). It should be noted that enzyme concentrations, as well as substrate type and concentration used in these calculation of dissociation constants varies among the different enzymes. 3.4.3 Chitin binding Unlike Cex, the fusion does not bind to chitin (See data Table X). No mention has been made in the literature of the ability of Cbhl to bind chitin. Figure 18 Adsorption data of Cex/Cbhl fusion on BMCC; graphed as a Lineweaver-Burke plot. 0 .5 0 . 0 1.0 2 . 0 3 .0 . . 4 . 0 5 .0 6 .0 1/F (L/nmole) 5 1 Table IX. Dissociation constants (Kd) (Stahlberg et al. 1991) and Kd/[FLS]i values for Cbhl, Cex and the Cex/Cbhl fusion protein. Enzyme Kd (uM) Kd/[FLS]i . . (g BMCC/L) Cex/Cbhl fusion - 0.066 Cex - 0.010 Cbhl 0.052 Cbhl CBD .0.480 -. 5 2 Table X. . Adsorption data for Cex/Cbhl fusion on chitin Sample# [P]o (mM) Free (mM) Bound (mmole/ g chitin) Bound/Free (mL/ mg chitin) •la 2.28e-4 1.92e-4 3.55e-5 0.184 lb 2.28e-4 2.04e-4 2.41e-5 0.118 2a 5.72e-4 5.27e-4 4.41e-5 0.084 2b 5.72e-4 5.27e-4 4.41e-5 0.084 3a 9.15e-4 8.39e-4 7.56e-5 0.090 3b 9.15e-4 9.22e-4 -7.14e-6 -0.008 4a 1.37e-3 1.31e-3 5.79e-5 0.044 4b 1.37e-3 1.33e-3 4.24e-5 0.032 5a 1.72e-3 1.60e-3 l;17e-4 0.074 5b 1.72e-3 1.64e-3 7.34e-5 0.045 6a 2.29e-3 2.27e-3 1.62e-5 0.007 6b 2.29e-3 2.23e-3 5.24e-5 0.024 7a 6.86e-3 6.79e-3 6.22e-5 0.009 7b ' 6.86e-3 7.19e-3 -331e-4 -0.046 5 3 3.5 Activity assays Preliminary assays were done to determine if the Cex/Cbhl fusion had catalytic activity on pNPL, C M C , xylan and BMCC. See Table XI for results and comparisons with Cex and Cbhl. 54 Table XI Activity assay results for Cex/Cbhl fusion. Cex and Cbhl activities are also summarized (From Gilkes et al. 1984b, van Tilbeurgh et al. 1982, Shoemaker et al. 1983a). A '+' indicated that the enzyme is active on the substrate, while a '-' indicates that the enzyme showed no activity on the substrate. Substrate Cex/Cbhl fusion Cex Cbhl pNPL + + + pNPC + + ' + CMC ' . + + Xylan + + Microcrystalline - - + cellulose • . •3 4. DISCUSSION 4.1 Polymerase chain reaction The use of polymerase chain reaction was a quick and simple method of constructing the Cex/Cbhl fusion. However, the obvious drawback of this approach is the possible misincorporation of bases during the PCR reaction. Misincorporated bases cannot be corrected by Taq polymerase since it has no 3' to 5' exonuclease activity. In order to avoid misincorporation, appropriate concentrations of dNTP's (10-50 |i.M each) must be used. A dNTP concentration which is too low (<1 p.M) has been found to cause misincorporation, whereas a high dNTP concentration (>1 mM) will allow mismatches to be extended more efficiently (Innis and Gelfand 1990). Although different groups reported mutation rates ranging from L i e - 4 nucleotides per cycle to 1.7e~4 nucleotides per cycle (or 1/200 after 30 cycles) (Tindall and Kunkell 1988, Saiki et al. 1988), other studies have found an average mutation rate of less than 5e~6 errors per nucleotide incorporated per cycle (Gelfand and White 1990). The major differences in these experiments were that those with lower mutation rates used lower dNTP and M g 2 + ion concentrations and higher annealing temperatures (55°C). Since my experiments were done using these conditions, I would expect to have similarly low mutation rates. Instead I found a mutation rate of 2.1e~4 nucleotides per cycle (1/80 after 2 runs of 30 cycles each). The reason for this high mutation rate is possibly the large number of cycles used. Mutation rates are usually calculated on the basis of data from a 30 cycle PCR. Almost all mutations were G to A transitions, one of the most common mutations found in PCR products (Innis and Gelfand 1990). Most of the mutations in the fusion 56 gene were in different locations, suggesting that the mutations occured late in the amplification process. It seems that although mutation rates are generally very low, PCR is still somewhat unreliable with respect to the fidelity of nucleotide incorporation. It is therefore wise to sequence the PCR product to ensure that no mutations have been introduced. This was especially important in my study because I was comparing the activities of the fusion protein to those of the intact proteins. To avoid sequencing the entire gene after each PCR reaction, one can amplify the segment of the gene which is to be changed by PCR, then sequence that small fragment and subclone it back into the original gene. The use of this strategy would eliminate the need for large amounts of sequencing, as well as ensuring that no mutations are present in the clone due to PCR misincorporation errors. 4.2 Protein leakage to supernatant The presence of the fusion protein in the supernatant liquid of cultures of E. coli containing pDDl is not unprecedented. This same phenomenon was described for CenA and Cex by Guo et al. (1988). They showed that the presence of these proteins in the supernatant was due to leakage from the periplasm rather than secretion or cell lysis. (3-lactamase, which is normally present only in the periplasm, was found in the supernatant, whereas glucose-6-phosphate dehydrogenase, a cytoplasmic enzyme, did not appear in the supernatant. It was suggested that the excess of protein present in the periplasm causes destabilization of the outer membrane (Gatz and Hillen 1986) and leakage of the periplasmic proteins. Since my protein carries the same signal sequence as Cex, it is likely exported to the periplasm and leaked into the supernatant in a similar manner. ' 4.3 Binding assays The analysis of cellulose binding by Erik Jervis takes into account the fact that some lattice sites may not be available for binding. This is a factor that previously has not been included in analyses of adsorption data. His analysis suggests that it is not possible to determine an absolute value for the dissociation constant, but rather a relative value. Therefore, this dissociation constant value cannot be compared to those of other enzymes unless they have been assayed using the same cellulose preparation. Using standard Scatchard analysis to determine the dissociation constant of my fusion protein, I found a Kd value of 0.0033 | iM. Although this value should not be used for the reason discussed, it is interesting that this value is much lower than the Kd values seen for either Cex or Cbhl. Both Cex and Cbhl were assayed using higher enzyme concentrations and using different types and concentrations of cellulose. A study of these three enzymes in the same concentrations, using the same substrate, and analyzed by the Lineweaver-Burke plot may provide some interesting information. It was interesting . to note that while my fusion protein reached maximum binding in less than one minute on BMCC, the literature shows that for a similar ratio of enzyme to cellulose, Cbhl takes up to 2 hours to reach the maximum bound on Avicel (Stahlberg et al. 1991). The differences between Stahlberg's experiments and mine include a difference, in the buffer used (citrate buffer p H 5 vs. potassium phosphate buffer p H 7), different concentration of substrates (10 or 50 mg/mL), different concentration of enzymes, and different substrates. Although both Avicel and BMCC are microcrystalline cellulose, Avicel, with a crystallinity index of 47% (Henrissat et al. 1985), is much less crystalline than BMCC. Stahlberg et al. (1991) noticed that the time required to reach maximum binding decreased with decreasing enzyme concentration. The lowest concentration they used was 0.5 nmole/mL which took over 10 minutes to reach 90% of the total amount bound. My highest protein concentration was very close to that at 0.55 nmole/mL but I found that the protein achieved maximum binding in less than one minute. Also, Stahlberg used a higher cellulose concentration than I did, so for the same concentration of enzyme, his experiments had 10 times as much cellulose. Still, he found longer binding times than I did. This difference could be due to any one of a number of factors, including the different substrates used, the difference in buffers, and possibly even the fact that intact Cbhl degrades Avicel while the fusion is unable to degrade BMCC. 4.4 Binding and catalytic specificities Binding and catalytic specificities are largely attributed to the binding and catalytic domains respectively. However, it is thought that these functional separations are not always clear. Thus, I was interested in investigating the specificities of the fusion protein. I found that although Cex can bind chitin, the fusion protein cannot. The literature does not state whether or not Cbhl can bind chitin, but it is unlikely since the fusion protein cannot. Like Cex, the fusion protein has the ability to degrade xylan and CMC, which Cbhl is not able to degrade. Also, again like Cex, the fusion protein is unable to degrade microcrystalline cellulose. 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