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Cloning and over-expression of processing a-glucosidase I gene from Saccharomyces cerevisiae (CWH41)… Dhanawansa, Ranjani 2004

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CLONING AND OVER-EXPRESSION OF PROCESSING cc-GLUCOSIDASE I GENE FROM Saccharomyces cerevisiae (CWH41) AND PURIFICATION OF THE SOLUBLE FORM OF THE ENZYME.  By RANJANI DHANAWANSA B.Sc. (Honours), University of Peradeniya, Sri Lanka, 1979 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE IN THE F A C U L T Y OF GRADUATE STUDIES (Food Science)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA SEPTEMBER, 2004 ©Ranjani Dhanawansa, 2004  DrTif-ii  F A C U L T Y OF G R A D U A T E S T U D I E S  T H E U N I V E R S I T Y OF BRITISH C O L U M B I A  Library Authorization  I  4* r  In presenting this thesis in partial fulfillment 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.  G.L.RANJANI DHANAWANSA  08/10/04  Name of Author (please print)  Date (dd/mm/yyyy)  Title of Thesis:  CLONING A N D OVER-EXPRESSION O F PROCESSING a-GLUCOSIDASE I G E N E FROM  Saccharomyces  cerevisiae  Degree:  M.Sc  Department of  FOOD S C I E N C E  (CWH4I> A N D P U R I F I C A T I O N O F T H E S O L U B L E F O R M O F T H E E N Z Y M E .  Year:  2004  The University of British Columbia Vancouver, BC Canada  grad.ubc.ca/forms/?formlD=THS  page 1 of 1  last updated: 7-Oct-04  ABSTRACT Processing a-glucosidase I (E.C. 3.2.1.106) is a key enzyme in the N-linked (asparagine-linked) oligosaccharide process pathway. It cleaves the oc-l,2-linked terminal glucose residue from the oligosaccharide precursor Glc3ManoGlcNAc2, which is crucial for the oligosaccharide maturation pathway. This pathway ultimately leads to the formation of complex, hybrid-type and high-mannose N-linked glycoproteins. Low abundance of the native enzyme is the major drawback in studying the mechanism and further characterization of glucosidase I. Preliminary trials were carried out for the isolation of the soluble fraction of the membrane bound processing glucosidase I from Saccharomyces cerevisiae. "Self-autolyzing" commercial dry yeast, purchased from Sigma Chemical Company, was used as the source of the enzyme. The yield and the activity of the isolated enzyme were insufficient for further studies. Therefore, over-expression of the gene encoding processing glucosidase I was the major objective of this research. In S. cerevisiae, the gene CWH41 encodes processing alpha glucosidase I. The open reading frame (ORF) of CWH41 was amplified with PCR (polymerase chain reaction) using genomic D N A from S. cerevisiae as the template. Primers were designed to introduce 5'Bglll and 3'XhoI restriction sites for directional cloning. The amplified PCR fragment was subcloned to a multicopy shuttle vector pHVX2 harbouring the constitutive phosphoglycerate kinase (PGK1) promoter/terminator cassette to yield the shuttle vector p R A N l . Using E. coli host DH5a, ampicillin resistant transformants were selected. The nucleotide sequence of the CWH41 was confirmed by sequencing the entire ORF. The predicted amino acid composition from the ORF matched that of the published amino acid sequence for CWH41.  ii  CWH41 was over expressed using S. cerevisiae host AH22. Yeast transformants of p R A N l and pHVX2 (control), selected for L E U 2 were used as the starting cultures for the isolation of the soluble fraction of the processing glucosidase I. The glucosidase I was extracted using glass bead cell disruption followed by centrifugation. The crude extracts were subjected to ultracentrifugation, ammonium sulfate precipitation, D E A E weak anion exchange chromatography, FPLC based Mono Q strong anion exchange chromatography and FPLC based Superdex 200 HR 10/30 gel filtration chromatography. Cloning, over-expression and partial purification of soluble processing glucosidase I was accomplished. Approximately a 30-fold increase in total enzyme activity of the recombinant clones compared to the control, after Mono Q anion exchange chromatography was noted. The glucosidase I active fraction eluted with Mono Q anion exchange chromatography showed a prominent protein band at 98 kDa on reducing SDS-PAGE. The glucosidase I active fraction eluted with gel filtration had a molecular weight of about 89 kDa. Expression of CWH41 in S. cerevisiae under the control of constitutive PGK1 promoter and terminator system (pRANl) provides an excellent system for overproduction of cc-glucosidase I for further studies.  iii  T A B L E OF CONTENTS ABSTRACT  ii  T A B L E OF CONTENTS  iv  LIST OF T A B L E S  vii  LIST OF FIGURES  ix  LIST OF A B B R E V I A T I O N S A N D S Y M B O L S  xi  ACKNOWLEDGEMENTS  xiV  PREFACE  xiii  1.0. INTRODUCTION  1  2.0. L I T E R A T U R E R E V I E W  3  2.1. Regulation of the biosynthesis of N-linked glycoproteins  3  2.2. Assembly of the lipid-linked core oligosaccharide  3  2.3. Significance of glucose addition  4  2.4. N-linked oligosaccharide processing  5  2.5. Processing alpha glucosidase 1  8  2.6. Inhibitors of glucosidase 1  13  2.6.1. Antiviral effects of glucosidase inhibitors  16  2.6.2. Anti-tumor effects of glucosidase inhibitors  18  2.7. Molecular cloning of processing glucosidase 1  19  2.8. Advantages of using yeast as a cloning vehicle  21 .  2.9. Use of S. cerevisiae constitutive expression vector PGK1  24  iv  3.0. M A T E R I A L S A N D METHODS  27  3.1. Materials  27  3.2. Microbial strains and plasmids  27  3.3. Media and growth conditions  28  3.4. D N A methodology  28  3.4.1. Preparation of competent E. coli  28  3.4.2. E. coli plasmid D N A extraction  28  3.4.3. E. coli tansformation of and selection of recombinants  29  3.4.4. Restriction endonuclease digestion of D N A  30  3.4.5. Agarose gel electrophoresis  30  3.4.6. PCR amplification and sub cloning  30  3.4.7. Ligation E. coli transformation and selection of recombinants  32  3.4.8. D N A sequencing and sequencing strategy  32  3.4.9. Yeast transformation and selection  34  3.5. Isolation of Soluble Glucosidase 1  34  3.5.1. Isolation of soluble glucosidase I from yeast suspension prepared from "self-autolyzing" yeast cells in 10 m M sodium phosphate buffer (pH 6.8)  34  3.5.2. Isolation of glucosidase from recombinant (pRANl) and native yeast (pHVX2) cultures  36  3.5.2.1. Growth of the culture  36  3.5.2.2. Glass bead cell extraction and centrifugation  36  3.5.2.3. Ammonium sulfate precipitation  38  v  3.5.2.4. D E A E -Sepharose anion-exchange chromatography  38  3.5.2.5. Ultrafiltration  39  3.5.2.6. FPLC with Mono Q HR 5/5, anion exchange column  39  3.5.2.7. FPLC with Superdex 200 HR 10/30, gel filtration column...40 3.6. Enzymatic assays  40  3.6.1. Glucosidase I activity assay  40  3.6.2. Aryl glucosidase activity assay  41  3.7. Determination of protein concentration  42  3.8. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)  42  4.0. RESULTS A N D DISCUSSION  44  4.1. Isolation of soluble glucosidase I from a yeast suspension prepared from "self-autolyzing" dry yeast cells  44  4.2. Cloning, isolation and characterization of E. coli clones harbouring processing glucosidase I gene from S. cerevisiae  53  4.3. Purification of soluble glucosidase I isolated from S. cerevisiae harbouring the plasmid p R A N l (recombinant) or p H V X 2 (control)  56  4.4. Gel filtration chromatography  70  4.5. Stability of the glucosidase I at 4°C for a period of 48 days  74  5.0. CONCLUSIONS A N D R E C O M M E N D A T I O N S FOR F U T U R E R E S E A R C H  76  6.0. REFERENCES  78  7.0. APPENDIX 1  94  8.0. APPENDIX II  98  vi  LIST OF T A B L E S Table  Page  1.  Microbial strains and plasmids used in this study  27  2.  Primers used for PCR and sequencing  31  3.  Purification of soluble glucosidase I isolated from "self-autolyzing" yeast suspension with one hour assay time  4.  Aryl glucosidase activity in fractions obtain from "self-autolyzing" yeast suspension  5.  51  52  Purification of soluble glucosidase I isolated from recombinant S. cerevisiae (pRANl) clones #6 and #12 grown in minimal media supplemented with L histidine with one hour assay time  6.  58  Purification of soluble glucosidase I isolated from control S. cerevisiae (pHVX2) grown in minimal media supplemented with L-histidine with one hour assay time  7.  59  Purification of soluble glucosidase I isolated from recombinant yeast clone #12 (pRANl) from S. cerevisiae grown in minimal media supplemented with L-histidine with one hour assay time  8.  65  Purification of soluble glucosidase I isolated from recombinant yeast clone #12 (pRANl) or the control (pHVX2) from S. cerevisiae grown in minimal media supplemented with L-histidine with 15 minute assay time  vii  67  Table 9.  Page Purification of soluble glucosidase I isolated from recombinant yeast clone #12 (pRANl) from S. cerevisiae grown in minimal media supplemented with L-histidine with one hour assay time  71  viii  LIST OF FIGURES Page  Figure 1.  Structures of glucosidase inhibitors  15  2.  Schematic summary of sequencing strategy of glucosidase I  33  3.  Soluble glucosidase I isolation scheme  37  4.  Reaction pathway for glucosidase I activity assay  43  5.  D E A E Sepharose elution profile of ammonium sulfate precipitated fraction of processing a-glucosidase I from "self-autolyzing" dry yeast.... 48  6.  The cloning strategy: CWH41 was cloned as a 2.5kb Bgl IVXhcA fragment into Bgl WXho\ sites of pHVX2 to yield p R A N l  7.  54  D E A E Sepharose elution profile of ammonium sulfate precipitated fraction of processing a glucosidase I from 63  Clone #12 of S. cerevisiae p R A N l 8.  F P L C anion exchange(Mono Q) chromatography of dialyzed, 0.3 M NaCl D E A E eluted fraction of the processing alpha glucosidase I from clone #12 of S. 64  9.  SDS-Polyacrylamide gel electrophoretic analysis of fractions obtained during purification of processing a-glucosidase I from pHVX2 (control or "C") and p R A N l (over-expressed sample or"S")  ix  69  Figure 10.  Page FPLC gel filtration (Superdex 200 HR10/30) chromatography of the processing alpha glucosidase I activity peak from D E A E Sepharose  11.  72  Stability of glucosidase I isolated from clone #12 of p R A N l with FPLC (Mono Q) anion exchange chromatography over a period of 48 days  74  x  LIST OF ABBREVIATIONS A N D S Y M B O L S AIDS  Acquired immunodeficiency syndrome  Amp  Ampicillin  Amp  R  Ampicillin resistance  ATCC  American type culture collection  Bp  Base pairs  BSA  Bovine serum albumin  CAST  Casternospermine  CAZY  Carbohydrate-active-enzymes  cDNA  Complementary D N A  Con A  Concanavalin A  CWH41  Gene encoding processing a-glucosidase in S. cerevisiae  DEAE  Diethylaminoethyl anion exchange resin  DEPC  Diethyl pyrocarbonate  dNJM  1-Deoxynojirimycin  DNA  Deoxyribonucleic acid  EDTA  Ethylene diamine tetra acetic acid  ER  Endoplasmic reticulum  FPLC  Fast protein liquid chromatography  HBV  Hepatitis B virus  HCMV  Human cytomegalovirus  fflV-1  Human immunodeficiency virus  kb  Kilo base (1000 bases)  xi  KDa  Kilo dalton(s)  LB  Lauri a broth  MCS  Multiple cloning sites  MWCO  Molecular weight cut off  NAPS  Nucleic acid protein services  NB-dNJM  N-butyl-deoxynojirimycin  NEM  N-ethylmalcimide  NM-dNJM  N-methyl-deoxynojirimycin  NN-dNJM  N-nonyl-deoxynojirimycin  ORF  Open reading frame  PAGE  Polyacrylamide gel electrophoresis  PCR  Polymerase chain reaction  PEG  Polyethylene glycol  PGK1  phosphoglycerate kinase  PMSF  phenylmethylsulfonyl fluoride  PNPG  p-nitrophenyl-alpha-D-glucopyranoside  SDS  Sodium dodecyl sulfate  TBE  Tris boric acid E D T A  TE  Tris E D T A  UBC  University of British Columbia  VSV  Vesicular stomatitis virus  UV  Ultraviolet  xii  ACKNOWLEDGEMENTS I would like to specially thank Dr. C. Seaman for her constant support, patience and encouragement throughout the course of my research. I am very grateful to the members of my research supervisory committee. Dr. E. Li-Chan, Dr. H . van Vuuren and Dr. R.A.J. Warren for their guidance and helpful suggestions during committee meetings and proof reading of this thesis. I also like to thank my family for their help. I appreciate the technical assistance given by Sherman Yee, Val Skura and George van der Merwe.  I dedicate this thesis to my parents.  xii/  1.0. I N T R O D U C T I O N Mannosyl oligosaccharide glucosidase (E.C.3.2.1.106), the first enzyme in the N linked (asparagine-linked) oligosaccharide-processing pathway, cleaves the distal a-1, 2linked glucose residue from the non-reducing end of Glc3ManaGlcNAc2 oligosaccharide precursor (Kilker et al, 1981; Bause et al., 1986). Subsequently, glucosidase II cleaves the two a-1, 3-linked glucose residues from the Glc2ManoGlcNAc2 oligosaccharide precursor. Then, up to four a-1, 2-linked mannose residues are removed by the endoplasmic reticulum (ER) resident and Golgi resident mannosidases (Lubas and Spiro, 1987; Tulsiani and Touster, 1988; Schweden and Bause, 1989). This pathway ultimately leads to the formation of complex and hybrid types of N-linked glycoproteins (Kornfeld and Kornfeld, 1985; Moreman et al, 1994). Asparagine-linked oligosaccharides have been implicated as playing an important role in many biological processes, such as cell growth, cell development and communication, as well as for protein stability and the control of protein folding (Varki, 1993; Parodi, 2000a). The cellular function of the transient glucose residues in the Glc3Man GlcNac2 9  precursor is unclear. A number of roles have been suggested for the glucose trimming reactions in mammalian cells. Possible roles include the mediation of lipid linked Glc3Manc;GlcNac2 transfer to the polypeptide chain (Spiro et al, 1979) as well as protection of the oligosaccharide precursor from degradation by phosphodiesterases (Hoflack et al, 1981). Some studies have demonstrated that blocking glucose removal with glucosidase inhibitors such as 1-deoxynojirimycin (dNJM) or castanospermine (CAST) causes the accumulation, and in some cases, degradation of some glycoproteins in the endoplasmic reticulum, thus decreasing their secretion and cell surface expression (Datema et al, 1987;  1  Elbein, 1991). The retention of glucose residues in glycoproteins can therefore be deleterious to intracellular transport through the secretory pathway. Glucosidase inhibitors can also act on the processing of some viral glycoproteins essential for the formation of the infectious virus particles. Therefore, glucosidase I inhibitors may be used as therapeutic agents against some viruses such as human immunodeficiency virus (HIV) and hepatitis B virus (HBV). The S. cerevisiae alpha 1,2-glucosidase I encoded by CWH41 is located in the endoplasmic reticulum. Since this enzyme plays such a key role in folding and transfer of N glycoproteins from the endoplasmic reticulum to Golgi, it might be expected to be a regulatory enzyme. Very little information is currently available regarding the mechanism of action of glucosidase I. The low abundance of this enzyme is a major drawback in carrying out further structural and mechanistic studies. Over expression of the gene encoding glucosidase I (CWH41) will help to produce sufficient protein to study structure and the mechanism of action of the enzyme. With this in mind, this research project was focused on cloning and over-expressing the gene encoding glucosidase I, and purifying the soluble fraction of enzyme recovered from yeast, S. cerevisiae.  2  2.0. LITERATURE REVIEW 2.1. Regulation of the biosynthesis of N-linked glycoproteins The subject of biosynthesis of N-linked oligosaccharides has been reviewed in detail (Kornfeld and Kornfeld, 1976; Hubbard and Ivatt, 1981; Schwarz and Datema, 1982; Kornfeld and Kornfeld, 1985). The biosynthesis process can be divided into three phases. (1) assembly of a high mannose oligosaccharide on the membrane-bound lipid, dolichol diphosphate (Hemming, 1985); (2) transfer of this oligosaccharide to protein, usually a nascent polypeptide chain; (3) processing of the protein-bound oligosaccharide to generate the complex, hybrid-type and high-mannose oligosaccharides by the action of glucosidases and glycosyl transferases (Beyer et ah, 1981; Schachter, 1984; Kornfeld and Kornfeld, 1985). An alternative processing pathway utilizes an endo-alpha-D-mannosidase to remove the Glc 1.3 Man fragment from newly glucosylated peptide (Lubas and Spiro, 1987). By removing a glucosylmannose disaccharide and yielding only one Man8GlcNAc isomer, this endo-alpha-D-mannosidase provides a processing route alternative to the sequential actions of alpha-glucosidase II and alpha-mannosidase I  2.2. Assembly of the lipid-linked core oligosaccharide Assembly of the lipid-linked core oligosaccharide Glc3ManoGlcNAc2 occurs via the dolichol cycle at the membrane of the rough endoplasmic reticulum and is a highly conserved process in eukaryotic cells (Kornfeld and Kornfeld, 1985; Tanner and Lehle, 1987; Herscovics and Orlean, 1993). This core oligosaccharide is formed by the sequential addition of the sugars N-acetylglucosamine (GlcNAc), mannose (Man) and glucose (Glc)  3  from their activated derivatives to the lipid carrier dolichyl-pyrophosphate ((Kornfeld and  Kornfeld, 1985; Verbert et ah, 1987; Abeijon and Hirschberg, 1992; Hirschberg and Snider, 1987; Herscovics and Orlean, 1993). The stepwise addition of three glucose residues (two oc1,3-linked glucose residues and a terminal oc-l,2-linked glucose residue) to dolichol-linked oligosaccharide is the final modification in the maturation of the carbohydrate substrate prior to transfer to protein. One exception to this was reported in trypanosomatids (Bosch et al., 1988; Parodi, 1993). In these organisms, the oligosaccharides transferred in vivo from dolichol-pyrophosphate derivatives lack glucose residues and only contain two N acetylglucoseamine and six, seven or nine mannose units depending on the species.  2.3. Significance of glucose addition The cellular function of the transient glucose residues in the Glc Man9GlcNAc2 3  precursor is unclear. Since glucose has not been found in carbohydrate units of glycoproteins containing mannose and N-acetylglucosamine, its presence in the lipid-saccharide donor may have a specific role. Turco et al. (1977) suggested that the attachment of glucose to mannosyl lipid-linked oligosaccharide served an important role in the transfer of these compounds from lipid carrier to protein acceptor. Spiro et al. (1979) reported that the presence of glucose in the oligosaccharide-lipid was essential for the transfer reaction to take place as removal of glucose by a specific thyroid glucosidase completely abolished donor activity. They also reported that the removal of peripheral mannose residues from oligosaccharide-lipid by a a-mannosidase digestion did not affect the transfer reaction. Burda and Aebi (1998) suggested that the terminal oc-l,2-linked glucose residue on the lipid  4  bound oligosaccharide serves as a signal to indicate complete assembly of the core oligosaccharide.  2.4. N-linked oligosaccharide processing Processing of the N-linked oligosaccharide begins immediately following the transfer of the Glc Man9;GlcNAc2 oligosaccharide precursor from its dolichol-bound form to a 3  specific asparagine residue of the nascent protein. Once the oligosaccharide is transferred to protein, and while the polypeptide is still being synthesized on membrane-bound polysomes, the oligosaccharide chain begins to undergo a series of processing reactions (Ugalde et al., 1980). a-Glucosidase I encoded by CWH41 cleaves the terminal a-l,2-linked glucose and ocglucosidase II encoded by ROT2 removes the two a-1, 3-linked glucose residues from the Glc3Man GlcNAc (Herscovics, 1999). Researchers who studied N-glycoprotein 9  2  biosynthesis in a variety of eukaryotic systems came into an agreement that the hydrolysis of the three glucose residues from the precursor oligosaccharide by glucosidase I and II is a universal occurrence in eukaryotes (Herscovics et al., 1977; Spiro et al., 1979; Ugalde et al., 1980 and Henner et al, 1981). Trimming of the glucose residues is identical in S. cerevisiae and in mammalian cells but a different number of mannose residues may be cleaved depending on the organism (Herscovics, 1999). In S. cerevisiae, only one specific a-l,2-linked mannose is removed in the endoplasmic reticulum (ER) with the formation of a single MangGlcNAc2 isomer. Then additional mannose residues are added by Golgi a-1,6-, a-1,2- and a-1,3-  5  mannosyltransferases to form mature high mannose oligosaccharide cores (Ballou, 1990; Herscovics and Orlean, 1993). In mammalian cells, different Man8GlcNAc2 isomers can be formed and additional a 1,2-mannose residues may be removed in the E R and the Golgi. Then, a variety of glycosyltransferases add different sugars, other than mannose, to form complex and hybrid N-glycans (Kornfeld and Kornfeld, 1985; Moreman et al, 1994). At present very little is known about the regulation of the biosynthesis of asparaginelinked glycoproteins, especially the reactions involving glucosidase I and glucosidase II. One thing all researchers agree is that all glycoproteins, whether high mannose, hybrid or complex type, must undergo processing by glucosidases. The catalytic action of these enzymes would appear to control overall biosynthesis of these proteins (Pukazhenthi et al, 1993a). The endoplasmic reticulum is the site where glycoproteins following the secretory pathway acquire their tertiary and in some cases, quaternary structures. When glycoproteins formed in the endoplasmic reticulum are misfolded, they are prevented from exiting to the Golgi apparatus. They are generally translocated into the cytosol for ubiquitination and are subsequently degraded by the proteasome. This system, the so-called ER-associated glycoprotein degradation, is important for eukaryotes to maintain the quality of glycoproteins generated in the ER (Herscovics, 1999). A mechanism for retaining improperly folded glycoproteins and facilitating acquisition of their native tertiary and quaternary structures operates in mammalian endoplasmic reticulum (Parodi, 2000b). In mammalian cells, the trimming of glucose is connected to a chaperone cycle involving two lectin-like chaperones, calnexin and  6  calreticulin. Calnexin is a type I membrane-bound molecule (Degen and Williams, 1991) and calreticulin a soluble luminal protein (Michalak et al, 1992). They specifically bind to monoglucosylated N-linked glycans (GlciMan5_9GlcNAc2) that are formed after removal of the two outermost glucoses by E R glucosidases or by the action of the UDP glucose: glycoproteinglucosyltransferase (UGGTase) (Hammond et al., 1994; Herbert et al., 1995; Peterson et al, 1995;Ware et al., 1995; Spiro et al., 1996). Calnexin binds transiently and selectively to proteins, anchoring them within the ER. This binding depends on the presence of a monoglucosylated oligosaccharide (Parham, 1996). Calnexin's binding is mediated by a lectin site recognizing the N-linked oligosaccharide processing intermediate GlciManaGlcNAc2 (Vassilakos et al., 1998). Calreticulin also interacts with monoglucosylated glycoproteins (Peterson et al, 1995; Otteken and Moss, 1996). No difference in glycoprotein binding was observed between calnexin and calreticulin in vitro (Vassilakos et al, 1998). However, the two lectins do not behave identically in vivo. It was reported that the patterns of glycoproteins precipitated with anti-calnexin and anticalreticulin antisera from lysed mammalian cells overlapped only partially (Peterson et al, 1995). UGGTase resides in the E R and adds back a single glucose residue to fully deglucosylated N-glycans (Trombetta et al, 1989). Since the glucosyl transferase only reglucosylates incompletely folded proteins, it is thought to serve as a folding sensor in the quality control process (Sousa et al, 1992; Hammond and Helenius, 1993). The addition and removal of glucose residues allows substrates to undergo cycles of binding and release with calnexin and calreticulin which promotes correct folding and at the same time prevents incompletely folded proteins from exiting the ER.  7  The lectin-monoglucosylated oligosaccharide interaction is one of the alternative ways by which cells retain improperly folded glycoproteins in the endoplasmic reticulum. Although it decreases the folding rate, it increases folding efficiency, prevents premature glycoprotein oligomerization and degradation, and suppresses the formation of non-native disulfide bonds by hindering aggregation (Parodi, 2000b).  2.5. Processing alpha glucosidase I Processing glucosidase I (E.C.3.2.1.106) belongs to the glycosyl hydrolase family 63 (http://afmb.cnrs-mrs.fr/CAZY/acc.html). It is a high mannose glycoprotein. Glucosidase I catalyzes the onset of processing reactions by trimming the terminal a-l,2-linked glucosyl residue even as early as when the polypeptide is still undergoing synthesis on the polysomes and translocating into the lumen of the endoplasmic reticulum (Atkinson and Lee, 1984). Spiro et al. (1979) first reported the occurrence of glucosidase I in calf thyroid microsomes. They also reported that the enzyme selectively releases glucose from dolichollinked oligosaccharide or from the oligosaccharide after transfer to the endogeneous protein. The unusual substrate specificity of the enzyme and its neutral pH optimum were two important characteristics they used to distinguish the enzyme from other thyroid glucosidases. However, they were not able to identify the a-glycosidic linkage of glucose residues and reported that glucosidase I acted on a-l,3-linkage. Kilker et al. (1981) partially purified glucosidase I from cell free extracts of yeast S. cerevisiae X-2180, using ammonium sulfate precipitation followed by DEAE-Sephadex chromatography. They reported that this enzyme removes glucose from non-reducing terminus of oligosaccharide precursor, Glc3ManoGlcNAc2. They also reported that the  8  enzyme was equally distributed between the particulate and the supernatant fractions obtained after centrifugation of the homogenate at 27,000g for 30 minutes. Membrane bound activity was stimulated by Triton X-100. The pH optimum of the enzyme was reported as 6.8. They also reported that this enzyme showed no requirement for divalent cations for activity. The addition of bovine serum albumin enhanced the activity. The glucosidase I activity was stable to lyophilization and unstable to dialysis. Hettkamp et al. (1984) purified glucosidase I from calf liver microsomes using affinity chromatography on AH-Sepharose 4B with N-carboxypentyl-l-deoxynojirimycin as ligand. They reported a subunit molecular mass of about 85 kDa by SDS-PAGE. They also reported the molecular mass of the native enzyme of 320-350 kDa by gel chromatography (Sephacryl S-300 column in 200mM phosphate pH 6.8 + 0.5% Lubrol P X as solvent) and suggested an association of subunits to a tetramer. The pH optimum of the calf glucosidase I was reported as 6.2. The enzyme was reported to be stable at 4 °C in the presence of detergent. Inhibition of this enzyme by 1-deoxynojirimycin with a median inhibitory concentration of 3 u M was reported. Bause et al. (1986) purified glucosidase I from yeast microsomal preparation by DEAE-Sephacel chromatography, affinity chromatography on AH-Sepharose 4B-linked N-5carboxypentyl-1-deoxynojirimycin and concanavalin A-Sepharose chromatography. They reported a subunit molecular mass of 95 kDa and confirmed that this glucosidase I was a glycoprotein. They also reported that this enzyme does not require any metal ions and the pH optimum was close to pH 6.8. Inhibition of yeast glucosidase I, by 2-dNJM, N-methyldeoxynojirimycin and N-5-carboxypentyl-deoxynojirimycin with JQ values of 16, 0.3 and 3 u M respectively, was reported.  9  Szumilo et al. (1986) purified the microsomal enzyme fraction from mung beans seedlings. The enzyme activity was released with 1.5% Triton X-100. They reported that the enzyme was purified 200-fold by chromatography on hydroxylapatite, Sephadex G-200, dextran-Sepharose and Concanavalin A-Sepharose. The pH optimum was reported as 6.36.5 and no requirement of divalent cations were observed. The inhibition of the enzyme by kojibiose in a dose-dependent manner was also reported. Strong inhibition by glucosidase processing inhibitors, casternospermine and deoxynojirimycin was also reported. Shailubhai et al. (1987) purified glucosidase I from lactating bovine mammary tissue. They solubilized the microsomal enzyme fraction with Triton X-100 and Lubrol P X and then subjected it to affinity chromatography on Affi-Gel 102 with N-5-carboxypentyl deoxynojirimycin as ligand and DEAE-Sepharose CL-6B chromatography. The molecular weight of the enzyme after SDS-PAGE under reducing conditions was reported as 85 kDa. A molecular mass of 320-330 kDa was reported after gel filtration and suggested that the native enzyme probably was a tetrameric protein. The pH optimum was reported as 6.6-7.0. They also reported that the enzyme did not require any metal ions for its activity. Inhibition of the enzyme by Hg , Ag and Cu 2+  +  2 +  and the reversal of inhibition by adding an excess of  dithiothreitol (DTT) was reported. Their results showed that kojibiose was an inhibitor of glucosidase I. They also raised rabbit polyclonal antibodies. Using Western-blot analysis and by immunoadsorption with Protein A-Sepharose, specificity of the anti-glucosidase I antibodies was confirmed. Bause et al. (1989) reported the purification (400-fold) and characterization of trimming glucosidase I from pig liver crude microsomes by fractional salt /detergent extraction, affinity chromatography and poly (ethylene glycol) precipitation. Molecular mass  10  of the enzyme was reported as 85 kDa. Binding to concanavalin A-Sepharose and its susceptibility to N-acetylglucosaminidase (endo H) led them to confirm that it was a N glycoprotein. The pH optimum of 6.4 was reported. They also reported that this enzyme did not require metal ions for activity and the native form of the enzyme is resistant to nonspecific proteolysis. They were able to cleave the enzyme with high (equimolar) concentrations of trypsin into protein fragments with molecular masses of 69 kDa, 45 kDa and 29 kDa and reported that it is composed of distinct protein domains. Strong inhibition by 1-deoxynojirimycin (Kj = 2.1 uM), N , N-dimethyl-1-deoxynojirimycin (K; = 0.5 uM) and N - (5-carboxypentyl)-l-deoxynojirimycin (K; = 0.45 uM) was reported. Presence of glucosidase I in rat brain was reported by Tulsiani et al. (1990). This finding was helpful in providing the evidence to support that the biosynthesis of N-linked glycoproteins was similar in rat brain and other tissues. Shailubhai et al. (1991) analyzed the functional domain structure of rat mammary glucosidase I. They suggested that the enzyme is a high mannose glycoprotein which can be cleaved by endo H . Based on trypsin digestion pattern and data on membrane topography, they reported that glucosidase I constitutes a single polypeptide chain of 85 kDa with two contiguous domains: a membrane-bound domain that anchors the protein to the endoplasmic reticulum and a luminal domain. They reported that a catalytically active 39-kDa domain could be released by limited proteolysis of saponin-permeabilized membranes with trypsin. Pukazhenthi et al. (1993a) studied the glucosidase I from several tissues of the rat, mouse, guinea pig and bovine mammary glands, sheep liver and pig kidney. They found that glucosidase I in all those tissues tested had a molecular weight of 85 kDa and it was cross reactive to anti-rat glucosidase antibody. The peptide mapping analysis of the enzyme from  11  different sources showed that major fragments of degradation of the enzyme from different sources were similar in size as determined by SDS-PAGE. Their results led to the conclusion that glucosidase I has been well conserved during the evolutionary development of the mammals. Another study done by Pukazhenthi et al. (1993b) reported the presence of one or more sulfhydryls and disulfide bonds in its primary structure of glucosidase. They also reported an involvement of - S H group in its activity. This critical - S H group was located at or near the catalytic site of the enzyme (Pukazhenthi et al, 1993b). Romaniouk and Vijay (1997) using a structure - function relationship study of glucosidase from bovine mammary gland reported that cysteine, arginine arid tryptophan were important amino acid residues in the catalytic site. They also reported that arginine and tryptophan residues play an important role in binding the substrate. Zeng and Elbein (1998) purified glucosidase I from microsomal fraction of mung bean seedlings. They reported that the enzyme had a subunit molecular weight of 97 kDa on SDS-PAGE gels and this mass was shifted to 94 kDa after treatment with Endo H or Endo F, suggesting that glucosidase I from mung beans was a glycoprotein with one oligomannosetype oligosaccharide. Glucosidase I activity was inhibited by kojibiose, Glc(ccl-2) Glc. Some similarities and differences between the plant and animal sources of the enzyme glucosidase I were also reported (Zeng and Elbein, 1998). The plant enzyme was inhibited by casternospermine and deoxynojirimycin. Yeast and mammalian processing glucosidase I were also inhibited by deoxynojirimycin (Saunier et al, 1982). The mung bean enzyme was quite sensitive to histidine modifying reagent diethyl pyrocarbonate (DEPC), whereas the pig liver glucosidase I was not. On the other-hand pig liver and pig brain  12  glucosidase I preparations were sensitive to the sulfhydryl reagent N E M (N-ethylmaleimide), whereas the plant enzyme was not sensitive. They reported that these sensitivities to amino acid modifiers suggest significant differences between plant and animal glucosidase I in terms of catalytic site or protein conformation. Using N M R spectroscopy, Palcic et al. (1999) reported that processing glucosidase I from yeast and bovine mammary gland are inverting hydrolases.  2.6. Inhibitors of glucosidase I Plant alkaloids block glycosylation by inhibiting the processing glycosidases involved in N-glycan formation. One such example is C A S T (1,6,7,8tetrahydroxyoctahydroindolizine). C A S T is isolated from the seeds of Australian plant Castanospermum australe (Hohenschutz et al, 1981). It inhibits both a-glucosidases I and II. This is a good inhibitor of the glucosidase I, since this alkaloid works best around pH 6.5 (Saul et al, 1984), which is close to the pH optimum (6.8) of glucosidase I. C A S T causes accumulation of fully glucosylated chains. Deoxynojirimycin (dNJM), is a glucose analogue with a N H group substituting for the O atom in the pyranose ring (Saunier et al, 1982). dNJM produced by Bacillus species, is the reduced form of norjirimycin, an antibiotic synthesized by Streptomyces. Other sugar analogues falling into this category are N-methyl deoxynojirimycin (NM-dNJM), /V-butyldeoxynojirimycin (NB-dNJM) and JV-nonyl- deoxynojirimycin (NN-dNJM) (Durantel et al, 2001). dNJM is a potent inhibitor of processing glucosidases (Saunier et al. (1982). The pH dependence of inhibition indicates that the cationic form of the inhibitors is the active species  13  and the electrostatic interactions at the catalytic site of the enzyme are important for inhibitor binding (Schweden et al, 1986). The reported concentration of dNJM needed for inhibition of glucosidase I and II were different for different organisms. Saunier et al. (1982) studied yeast enzymes and reported a 50% inhibition of glucosidase I (20 u M inhibitor) and glucosidase II (2 u M inhibitor). Hettkamp et al. (1984), using calf liver microsomes reported a 50% inhibition of glucosidase I (3 u M inhibitor) and glucosidase II (20 u M inhibitor). It was also reported that some of the reported biological effects of 1-dNJM are not necessarily related to the processing glucosidases and could be due in part to a general inhibition of the synthesis of lipid-linked oligosaccharides (Datema et al, 1984). N M - d N J M is produced by methylation of dNJM. The presence of a methyl group in this compound has two very important biological effects. As mentioned earlier, dNJM under certain conditions inhibits the synthesis of lipid-linked oligosaccharides (Datema et al, 1984; Romero et al, 1985). This inhibition is not observed in N M - d N J M treated cells (Romero et al, 1985). It was also reported that N M - d N J M is a more potent inhibitor of glucosidase I (Romero et al, 1985; Hettkamp et al, 1984). Romero et al. (1985) reported that N M - d N J M inhibits the synthesis of N-linked complex oligosaccharides in rat intestinal epithelial cells to the same extent as dNJM. They indicated that both inhibitors cause the accumulation of a mixture of glucosylated oligosaccharides containing one to three glucose residues and seven to nine and even possibly six, mannose residues. They also reported that about 70% of the endo H-sensitive oligosaccharides formed in the presence of N M - d N J M contained three glucose residues, compared with only about 20% of the corresponding oligosaccharides of the dNJM treated cells. Therefore, N M - d N J M is a better inhibitor for glucosidase I.  14  Castanospermine (CAST)  1-deoxynojirimycin (dNJM)  N-methyl-deoxynoj irimycin (NM-dNJM)  Figure 1. Structures of glucosidase inhibitors  15  High-resolution structures of the glucosidase inhibitors dNJM and C A S T have been determined by X-ray diffraction (Hempel et al, 1993). The absolute configuration of C A S T has also been established. In stereochemical comparisons with natural glucosidase substrates such as maltose and methyl glucoside, they reported that C A S T has similarities in the positioning of functional groups providing the basis for enzyme inhibition. After a conformational comparison between dNJM and CAST, the greater activity of C A S T was reported. The fixed axial positioning of the 0 6 atom in C A S T was reported as the reason for greater activity.  2.6.1. Antiviral effects of glucosidase inhibitors The outer envelope of many animal viruses is composed of one or more viral glycoproteins. These glycoproteins play an essential role in completing different stages of the viral life cycle such as virion assembly, secretion and infectivity. Inhibitors of glycoprotein processing pathway have been used to study the role of N-glycans in several viral systems. Some of these systems are human immunodeficiency virus (HIV-1) (Karpas et al, 1988), human hepatitis B virus (HBV) (Block et al, 1994), human cytomegalovirus (HCMV) (Taylor et al, 1988), influenza (Datema et al, 1987), Sinbis virus (Schlesinger et al, 1985) and vesicular stomatitis virus (VSV) (Schlesinger et al, 1984). Human immunodeficiency virus (HIV-1), the causative agent of acquired immunodeficiency syndrome (AIDS), produces two envelope glycoproteins (gpl20 and gp41) through endo-proteolytic cleavage of a precursor protein (gpl60) within the cis-Golgi apparatus (Mehta et al., 1998). After this cleavage, gpl20 remains non-covalently attached to the luminal portion of the transmembrane gp41 through conserved regions within the  16  amino and carboxy terminus. An interaction between the viral envelope gpl20 and CD4 protein is required to initiate an infectious cycle (Gruters et al 1987). During infection, gpl20 binds to the CD4 surface antigen and undergoes a conformational change and cleavage, which exposes gp41. The exposure of gp41 allows fusion with the cellular membrane, thus mediating viral entry into the cell (McCune et al., (1988). HIV is a retrovirus containing R N A . In order to reproduce, the virus must attach to a cell of the infected organism, insert its R N A into the cell and make D N A copy of the R N A . Data from three independent groups, Walker et a/.(1987), Gruters et al. (1987) and Tyms et al. (1987), suggested a potential role for glucosidase I inhibitors as anti-human immunodeficiency viral (anti-HIV) agents. Gruters et al, (1987) investigated the effects of CAST, dNJM and 1deoxymannojirimycin (dMJM), three processing glycosidase inhibitors which mimic N linked glycan structure, on induction of the formation of syncytium between HIV-infected and CD4-expressing cells. They reported that glucosidase inhibitors C A S T and dNJM, but not the mannosidase inhibitor dMJM, inhibited syncytium formation and interfered with infectivity. Sunkara et al. (1990) also repored the potential use of C A S T analogues as antiHIV agents. Fisher et al. (1995) reported that the treatment of HIV-1 infected cells with N B dNJM inhibits syncytium formation and the formation of infectious virus. Hepatitis B virus (HBV) is a causative agent of acute and chronic liver disease (Ayoola et al, 1988). The H B V envelope contains three co-carboxyl-terminal proteins (HBs proteins). A l l three HBs proteins have complex-type N-linked oligosaccharides (Block et al, 1994). Removal of terminal glucose from immature glycoproteins is thought to play an  17  important role in the migration of the these glycoproteins from E R to the Golgi ((Datema et al, 1987). The morphogenesis of H B V is complex. Pre-assembled viral core particles are believed to attach to the cytosolic side of viral envelope (surface) proteins, which have inserted into the endoplasmic reticulum (ER) membrane (Ganem, 1991). After acquiring envelopes, virions bud to the lumen of ER, from which they are transported through Golgi apparatus into the extracellular fluid (Heerman and Gerlich, 1991). Zitzmann et al. (1999) reported NN-dNJM-induced misfolding of one of the H B V envelope glycoproteins and prevented the formation and secretion of virus in vitro. They also reported that this inhibitor alters glycosylation and reduces the viral levels in an animal model of chronic H B V infection. In this virus, correct glycosylation appears to be necessary for processes involved in transport of the virus out of the host cell. In vitro treatment of H B V with N B - D N J results in a high proportion of virus particles being retained inside the cells (Block etal, 1994) Some problems still remain to be resolved in the use of processing glucosidase I inhibitors as anti-viral agents (Asano, 2003). Problems exist in achieving the therapeutic serum concentrations of inhibitors needed to inhibit glucosidase I (Asano, 2003). Side effects such as diarrhea due to non-specific inhibition of other glucosidases, is another problem (Cook et al., 1995).  2.6.2. Antitumor effects of glucosidase inhibitors C A S T has been shown to prevent the normal glycosylational processing of the v-fmstransforming glycoprotein (oncogene). These v-fms-transformed cells grown in vitro in the  18  presence of C A S T accumulate immature forms of the glycoprotein that do not reach the cell surface. These treated cells revert to the normal phenotype. Ostrander et al. (1988) extended those studies to an in vivo tumorigenicity mode in the nude mouse using v-fms-transformed rat cells (SM-FRE) and dietary administration of CAST. Their results suggested that these mice had slower growing tumors that were 2.6 times smaller than tumors in control groups at the termination of the experiment, 24 days post-injection. These results indicate that C A S T is effective in slowing the growth of v-fms-transformed cells in vivo, suggesting that this drug may offer an effective therapy against certain tumors, including some arising from activated protooncogenes that encode glycoproteins such as growth factor receptors. The development of drugs that target the tumor neovasculature may hold promise in inhibiting tumor growth. Endothelial cell proliferation, invasion of basement membrane, and differentiation are crucial steps during neovascularization. Using in vitro differentiation models of Matrigel and postconfluent cultures of endothelial cells, Pili et al. (1995) studied the effects of C A S T on endothelial cell behavior. Upon treatment with CAST, an increase in high mannose groups and a decrease in tri- and tetra antennary beta-linked galactose-Nacetylglucosamine on mannose residues of asparagines-linked oligosaccharides were reported. These studies suggested that certain cell surface oligosaccharides are required for angiogenesis and that glucosidase inhibitors that alter these structures on endothelial cells are able to inhibit tumor growth.  2.7. Molecular cloning of processing glucosidase I Kalz-Fuller et al. (1995) screened a human hippocampus cDNA library against oligonucleotide probes generated by PCR. Primers were derived from the amino acid  19  sequences of tryptic peptides of pig liver glucosidase I. Using 2 isolated clones, they constructed a full-length glucosidase I cDNA (2881 bp). They reported that this cDNA construct encoded a single open reading frame of 834 amino acids corresponding to a molecular mass of 92 kDa. Presence of one glycosylation site at Asn 655 was also reported. The glucosidase I c D N A construct was transfected into Cos 1 cells and overexpressed the enzyme activity about four fold. Oligonucleotide sequence showed no homology to other processing enzymes cloned to that time (Kalz-Fuller et al., 1995). The enzyme activity was strongly inhibited by 1-dNJM or N M - D N J M . The expressed enzyme with a molecular mass of 95 kDa was degraded by endoglycosidase H to a 93-kDa form. Proteolytic studies and immunofluorescence microscopy showed that overexpressed enzyme was a type-II transmembrane glycoprotein with a short cytosolic tail of about 37 amino acids, followed by a single transmembrane domain and a large C-terminal catalytic domain located on the luminal side of the endoplasmic reticulum membrane (KalzFuller et al, 1995). By fluorescence in situ hybridization, Kalz-Fuller et al. (1996), mapped the glucosidase I gene. The gene was located on chromosome 2. The localization was confirmed with PCR-based analysis of somatic cell hybrids. Using pRS316 based yeast genomic D N A library, Jiang et al.(1996) cloned the gene encoding membrane bound glucosidase I (CWH41) in S. cerevisiae The centromeric vector pRS316 was used to subclone the gene. The 2(xm based vector Yep351, was used to overexpress the native CWH41 gene (Jiang et al, 1996). They reported that the CWH41 gene is involved in assembly of cell wall 3 1,6 glucan, and encodes a type II integral membrane N-glycoprotein located in ER. Using D N A sequence analysis studies these researchers claimed that this cloned fragment has a single, 2.5-kb open reading frame  20  encoding a protein of 833 amino acid residues. They reported that cwh41p has a positively charged N-terminal tail of 10 amino acid residues followed by a stretch of 16 hydrophobic residues (potential transmembrane domain) and a large 807 amino acid C-terminal domain containing four potential glycosylation sites (Asn-x-Ser/Thr). The location of CWH41 gene was reported as the left arm of the chromosome VII, adjacent to the gene TRP5. Work by Romero et al. (1997) showed Cwh41p had a significant amino acid similarity to the product of the human glucosidase I cDNA. Tetrad analysis for glucosidase I activity in vitro and in vivo was done on the progeny of spores obtained from the heterozygous diploid, cwh41A::HIS3. They showed that cell extracts obtained from cwh41 delta null mutants were unable to release glucose residues from the synthetic trisaccharide substrate a-D-Glc (l-»2) a-D-Glc (l->3) a-D-Glc-0 (CH ) C O O C H 2  8  3  in vitro. Following  one hour labeling of cells with [ H] mannose and analysis by high pressure liquid 3  chromatography of the labeled N-linked oligosaccharides, combined with treatment with jack bean alpha mannosidase and yeast glucosidase I, they showed that the oligosaccharides isolated from a cwh41 delta null mutant were fully glucosylated. They also reported that the oligosaccharide from the null mutant retained the three terminal glucose residues and the oligosaccharides from CWH41 cells did not have any glucose residues. These results showing a lack of glucosidase I activity in cwh41 delta null mutants both in vitro and in vivo were consistent with the structural evidence that CWH41 encodes the yeast glucosidase I.  2.8. Advantages of using yeast as a cloning vehicle Yeast is the favoured alternative host for the expression of foreign proteins for research, industrial and medical use (Hitzeman et al., 1981). As a food organism, it is  21  acceptable for the production of pharmaceutical proteins. In contrast, E. coli has toxic cell wall pyrogens and mammalian cells may contain oncogenic or viral D N A (Hitzeman et al., 1981). S. cerevisiae expression system has advantages over bacterial and mammalian systems. Yeast cells are eukaryotic and are more similar to mammalian cells than a prokaryotic bacterial cell. Although high levels of transcription and translation of foreign genes can be accomplished in prokaryotic systems, such as Escherichia coli, the protein is deficient in a number of features. The products generally lack disulfide bonds and modified N-termini. Furthermore, post-translation modifications such as glycosylation do not occur in bacteria. Initial stages of post-translational glycosylation in the endoplasmic reticulum involving the trimming of glucose residues are identical in S. cerevisiae and in mammalian cells (Herscovics et al, 1977; Spiro et al, 1979; Ugalde et al, 1980 and Henner et al, 1981). Therefore, yeast is an excellent organism to study the initial stages of post translation glycosylation. Thus, eukaryotic systems such as S. cerevisiae have been utilized to express proteins. S. cerevisiae expresses intracellular proteins at a high level, while extracellular expression has been less successful. Yeast can be cultured like bacteria in flasks, whereas mammalian cells require meticulous care in tissue culture. Furthermore, the growth rate of yeast is much faster than mammalian tissue cultures, so a large cell mass can be grown faster and more cost effectively. This makes scale up much simpler than for mammalian cell culture. Yeast grows quickly in defined medium, and is easier and less expensive to work with compared to mammalian cells. When required, yeast expression systems are adaptable to large-scale production of the recombinant eukaryotic protein.  22  The yeast S. cerevisiae has become the most useful eukaryotic model for recombinant D N A technology. One of the main reasons is that the transmission genetics of yeast is extremely well understood. Another advantage of S. cerevisiae is its growth rate and simple nutritional requirement, which can be met with a minimal medium. A minimal medium for such yeast can contain glucose as the sole source of carbon. Metabolism of glucose to smaller molecules (e.g., CO2, ethanol, or acetic acid) can generate the A T P necessary for energy-requiring activities of the cells. The sole nitrogen source in a minimal medium can be ammonium (NH4 ), from which the cells can synthesize all the necessary amino acids and +  other nitrogen-containing metabolites. Salts and trace elements are the only other components of a minimal medium. Yeast, which grows in nature as single cells, is easily grown in culture dishes usually on top of agar. Usually a dilute suspension of cells is dispersed on top of the agar and each cell grows into a discrete colony. Since the cells in a colony all derive from a single cell, they form a clone and have identical D N A . A l l the cells in a clone generally express the same set of genes and contain the same enzymes and other constituents in similar proportions. E. co/z/yeast shuttle vectors are very useful in the routine cloning and manipulation of yeast genes (Griffiths et al, 1999). The simplest yeast vectors are derivatives of bacterial plasmids into which the yeast locus of interest has been inserted. Generally, these plasmid vectors contain genetic material derived from E. coli pBR322 (or its derivatives) and a genetic element (origin of replication), which enable them to be propagated in E. coli cells (prior to transformation into yeast cells) and a selectable marker (mainly the (3-lactamase gene, amp) for the bacterial host. When transformed into yeast cells, these plasmids insert  23  into yeast chromosomes generally by homologous recombination with the resident gene. As a result, either the entire plasmid is inserted or the targeted allele is replaced by the allele on the plasmid. Such integrations can be detected by plating cells on a medium that selects for the allele on the plasmid. Because bacterial plasmids do not replicate in yeast, integration is the only way to generate a stable modified genotype with the use of these vectors. In yeast, another important advantage is the availability of a circular 6.3-kb natural yeast plasmid. This plasmid, which has a circumference of 2 jum, has become known as the "2-micron" plasmid. It forms the basis for several sophisticated cloning vectors (Ludwig and Bruschi, 1991). The 2-jum segment has the ability to replicate autonomously in the yeast cell, and insertion is not necessary for a stable transformation. Second, genes can be introduced into yeast, and their effects can be studied in that organism; then the plasmid can be recovered and put back into E. coli, provided that a bacterial replication origin and a selectable bacterial marker are on the plasmid (Griffiths et al, 1999).  2.9. Use of 5. cerevisiae constitutive expression vector PGK1 Several well-characterized yeast expression systems, both inducible and constitutive, are available (Burgers, 1999). G A L (galactose), MET3 (methionine) and the CUP1 (metallothionein-in response to copper) are well known inducible promoters. ADH1 (alcohol dehydrogenase), PGK1 (phosphoglycerate kinase), G A P D H (glyceraldehydes - 3 - phosphate dehydrogenate) and ENO (enolase) are well known constitutive promoters. Constitutive promoters are derived from genes of the glycolytic pathway, because these lead to high-level transcriptional expression of the gene of interest.  24  Chen and Hitzeman (1987) studied the S. cerevisiae, PGK1 promoter in detail. They reported that when the gene encoding PGK1 is present on a high copy number plasmid, 3040 percent of yeast protein is PGK1. However, when the structural part of this gene was replaced by as many as twenty different heterologous genes, the production of gene products were reduced by more than 20 fold. They reported that this decrease in protein production is accompanied by large decreases in the steady-state levels of mRNA. They also reported that the replacement of the yeast P G K structural gene with a human P G K c D N A has little effect on the steady-state mRNA level in yeast (Chen and Hitzeman, 1987). Van der Aar et al. (1992) studied the effects of phosphoglycerate kinase (PGK) overproduction on the physiology and plasmid stability in S. cerevisiae containing the PGK1 gene on an episomal plasmid. They reported that there is a preferred intracellular level for this enzyme P G K 1 , amounting to 10-15% of the total soluble protein. Plasmid-containing strains and the host were grown in non-selective batch cultures and continuous cultures, under different growth conditions. They reported that plasmid-containing yeast strains stabilize the copy number of the episomal plasmid at a level at which the P G K concentration is about 12%. They also reported that PGK1 gene could be applied as a direct positive selection marker to obtain a high episomal plasmid stability during growth on glucose. Mellor et al. (1985) used the promoter fragment from P G K gene to direct the expression of human interferon-alpha-2 (IFN alpha 2) on a high-copy-number plasmid in yeast. The yields of IFN alpha 2 was only 1-3% of yeast total protein, whereas the maximum yield of P G K produced by the P G K gene on a high-copy-number plasmid was at least 50%. They reported that IFN alpha 2 is turned over more rapidly than P G K . They also reported  25  that the major reason for the relatively low level of IFN alpha 2 is that IFN-specific R N A levels were much lower. From these results, they concluded that the presence of heterologous coding sequences, or the absence of specific yeast sequences causes a reduction in heterologous R N A levels in yeast. Levels of P G K mRNA can sometimes be elevated by heat shock of yeast cultures. Piper et al. (1988) studied how specific deletions of P G K promoter sequences affect levels of P G K mRNA both before and after heat shock. It is proposed that PGK1 may be one member of a small subset of yeast genes that are highly expressed in unstressed cells yet possess a heat shock element to ensure their continued transcription after heat shock (Piper et al., 1988). Therefore, P G K mRNA is both elevated in level in response to heat-shock and translated efficiently at supra-optimal temperatures (Piper et al, 1986).  26  3.0. MATERIALS AND METHODS 3.1. Materials Nano-pure water (Millipore) was used in all experiments. Lab chemicals were of Molecular Biology grade and Electrophoresis grade except where noted. Restriction enzymes were purchased from Boehringer Mannheim, Gibco-BRL, New England Biolabs or Roche Molecular Biochemicals.  3.2. Microbial strains and plasmids The microbial strains and plasmids that were used in the present study with their relevant genotypes and sources are listed in Table 1. Plasmid constructed during the course of this work will be described in the results.  TABLE 1. Microbial strains and plasmids used in this study  Genotype  Source  mat a, leu 2-3, leu 2-112, his 4-519, canl  A T C C #38626  F7endAl,hsdRl 7( ri ), supE44,thilrecAl gyrA(Nal') relAlA(lacZYA-argF) UI 69 (m80lacZAM15)  Promega Co.  Plasmids Yep 351 + CWH41  Ap LEU2  Jiang et al., 1996  PHVX2  AP L E U 2 PGK1  Strain Yeast strain S. cerevisiae AH22 Bacterial strain E. coli strain DH5-0C  R  R  27  p t  Volschenk etal, 1997  3.3. Media and growth conditions The bacterial strain E. coli DH5a was used as the host for propagation of plasmids. This bacterial strain was routinely cultured in L B (lOg Bacto-tryptone, 5g Bacto-yeast extract, lOg sodium chloride per liter) liquid medium. For solid media 2% agar was added to L B liquid medium before autoclaving. Ampicillin (100 ixg/ml) was added to L B media and plates when growing bacteria containing plasmids. S. cerevisiae strain AH22 was used as the host and the strain was routinely maintained in Y E P D (1% Yeast Extract, 2% Peptone and 2% Dextrose). Transformants were maintained in sterile minimal media (0.67% Yeast Nitrogen Base without amino acids + 2% Dextrose) supplemented with filter sterilized L-histidine (50 |0,g/ml). For solid media 2% agar was added to minimal media before autoclaving.  3.4. DNA Methodology 3.4.1. Preparation of competent E. coli E. coli DH5a was made competent for transformation by the rubidium chloride method (Maniatis et al, 1982). Aliquots of competent cells (200 ul each) were distributed in sterile micro centrifuge tubes, flash frozen in liquid nitrogen, and stored at -80 °C.  3.4.2. E. coli plasmid DNA extraction Smaller scale D N A extractions were carried out using the procedure of Morelle (1987). This procedure routinely yielded 2-3 ixg of plasmid D N A pure enough for transformation reactions and for rapid analysis of recombinant clones with restriction enzyme digestion. The cells from 2.0 ml of saturated overnight cultures were used to extract  28  plasmid D N A . Clean pellet was air dried after precipitation with isopropyl alcohol and resuspended in 50 ul of sterile Tris E D T A (TE), pH 8.0. Contaminating R N A was removed by incubating the D N A samples with RNAse A for 30 minutes at 37 °C. The clean D N A was stored at -20°C and used as needed. Larger scale plasmid D N A extractions were carried out using P E G precipitation procedure (Maniatis et al. (1982). The cells from 500 ml of saturated overnight cultures were used to extract plasmid D N A . Quantity and purity of the D N A were determined by measuring the absorbance at 260 nm/280 nm ratio (1 OD 260 nm = 50 Ug/ml DNA). The samples with 260/280 ratio of 1.8-1.9 were diluted to 1 mg/ml with sterile T E (pH 8.0) or sterile water (sequencing templates) and stored at -20 °C.  3.4.3. E. coli transformation and selection of recombinants The E. coli transformation was carried out using the procedure described by Maniatis et al. (1982). For each transformation, 200 p,l of competent cells were used. Frozen cells were thawed on ice and mixed with 5 \x\ of purified plasmid D N A . The mixture was incubated for 45 minutes on ice, heat shocked for two minutes at 37 °C and placed back on ice. After two minutes, 800 ul of L B was added to the mixture. The contents were mixed gently and incubated for another 30 minutes at 37 °C. An aliquot (100 ul) of the mixture was plated on L B + Amp.(100 |xg/ml). The remainder of the cells was centrifuged for 10 seconds and 800 ui of the supernatant was removed. The pellet was re-suspended and plated on another L B + Amp (100 u.g/ml) plate. Plates were incubated overnight at 37 °C. A control sample without D N A was also transformed to check the efficiency of transformation. The plates with transformants were wrapped with parafilm and stored at 4 °C.  29  3.4.4. Restriction endonuclease digestion of DNA Restriction endonucleases were obtained from New England Biolabs or Gibco B R L and were used with the appropriate buffers by the supplier. The digestions were performed according to the manufacturer's specifications. Total volume for a single enzyme digestion was 10 ul and the incubations were for one hour at 37 °C. Total volume for a double enzyme digestion was 20 u.1 and the incubations were carried out overnight at 37 °C. A l l digests were monitored by agarose gel electrophoresis.  3.4.5. Agarose gel electrophoresis D N A fragments and restriction digestion mixtures were separated by agarose gel electrophoresis (Sambrook et al., 1989). 0.8-1.0% agarose (Gibco BRL;Ultrapure grade) gels were prepared in 1 x Tris Boric acid E D T A (TBE) (Appendix I). Ethidium bromide (0.7|ag/ml) was added to agarose just before pouring the gel. Gels were run in 1 x T B E at 50V constant voltage for 1-3 hours as required for sufficient separation of fragments. Migration of D N A was estimated by tracking dye in 2 x T B E sample buffer (Appendix I). D N A was visualized using a U V trans-illuminator at 254 nm and photographed. When needed desired fragments were cut and purified using Qiagen gel extraction columns (Qiagen Inc., Canada), following manufacturer's instructions.  3.4.6. PCR amplification and subcloning An open reading frame (ORF) of the gene CWH41 with added restriction sites BglR (5') / Xhol (3'), was amplified from genomic D N A using PCR, following manufacturers instructions. The PCR primer sequence is presented in Table 2.  30  Amplified PCR fragment was gel purified using Qiagen purification column. The purified fragment was digested overnight with BgllVXhol restriction enzyme mixture. Digested fragment was also cleaned with Qiagen column and stored at -20°C. Plasmid pHVX2 with P G K l  p t  cassette (Table I) was digested overnight with BglR  and Xhol enzyme mixture, gel purified and used as the parent vector to subclone the CWH41 gene. Purified fragment and the vector were subjected to agarose gel electrophoresis to check the purity and the concentration.  Table 2. Primers used for P C R and sequencing  Primer  Sequence  RDl  1,2  5'-AAT T A G A T C T C G G T T C A G GTT GCT T C A C A A - 3 '  RD2  1  5'-AAT TCT C G A G T C T A C A T A G A T T C A G A A GCG-3'  Rev20g  2  5' - A C A A G C A A T C G A A G G TTC T G - 3 '  RSP1  2  5' - T A A A G A A T T A G G C G A G T A TC -3'  RSP2  2  5' - A G T A T G A T T T T G A C C T T G C C -3'  RSP3  2  5'- A T C T G G A T T TGT T A C A C T G G -3'  RSP4  2  5'- A A T C G C T A A C T A C A G A T T G G - 3 '  'PCR primer; Sequencing primer  31  3.4.7.  Ligation, transformation of E. coli and selection of recombinants Ligation reactions were started with different molar ratios (Vector: Fragment ratio  1:0; 1:1; 1:2: 1:3). The total reaction volume was 50 ul. EnzymeT DNA Ligase (Gibco 4  BRL) with its own buffer was used. The ligation reactions were carried out overnight at 16 °C. Each reaction mixture was diluted five times before transformation. The E. coli transformation of the ligation reactions was carried out using the procedure described in Section 3.7. An aliquot of ligation mixture was substituted for D N A . E. coli clones harbouring recombinant plasmids were selected on LB-Amp. Agar plates incubated at 37 °C. Positive clones were stored either as single colonies at 4 °C or 20% glycerol stocks at -80 °C.  3.4.8.  DNA Sequencing and sequencing strategy Purified PEG D N A from Section 3.4.2 was used as the template for sequencing.  Primers were prepared in the Nucleic acid protein services (NAPS) unit, University of British Columbia. The nucleotide sequence of the primers is presented in Table 2. The sequences of the primers were based on the nucleotide sequence of the gene CWH41. The nucleotide sequence of the open reading frame of the gene CWH41 was confirmed with nucleotide sequence analysis of p R A N l . The details of the sequencing strategy are presented in Figure 2. Sequencing was carried out using the automated fluorescent dye terminator technique (Applied Biosystems model 377) in the NAPS Unit, University of British Columbia. The sequence results are presented in the Appendix II.  32  ATG  TGA j  CWH41 1000  3502  Rev20g  RD1  RSP1  ^ RSP2  RSP3 RSP 4  Primer  Number of bases sequenced  Rev20g  570  (1000-1570)  RD1  330  (1350-1680)  RSP1  748  (1652-2400)  RSP2  829  (2351-3180)  RSP3  460  (3042-3502)  RSP4  470  (2000-1500)  Figure 2. Schematic summary of sequencing strategy of glucosidase I  33  3.4.9. Yeast transformation and selection Yeast transformation was carried out using plasmid D N A from plasmids p R A N l and p H V X 2 . The lithium chloride method (Ito et al., 1983) was used. The yeast strain A H 2 2 ( A T C C #38626) was used as the host. A s a carrier, 5 m l of sheared salmon sperm D N A was added to the competent cell mixture. The transformants were spread on agar plates containing minimal media supplemented with filter sterilized L-histidine and incubated for 2-3 days. The plates with transformants were sealed with parafilm and stored at 4 °C.  3.5. Isolation of soluble glucosidase I 3.5.1. Isolation of soluble glucosidase I from a yeast suspension prepared from "selfautolyzing" yeast cells in 10 mM sodium phosphate buffer (pH 6.8). A known weight (50 g) of dry yeast cells ("self-autolyzing" dry yeast, SigmaY3502) were mixed with 200 m l of buffer A (10 m M sodium phosphate, p H 6.8) and allowed to form a cell suspension. The cell suspension was stirred for about half an hour at room temperature and then cooled to 4°C. Subsequent manipulations were carried out at 4 °C. Phenylmethylsulfonyl fluoride ( P M S F ) at 50 u M was added to the mixture, which was then passed twice through a pre-cooled French press at 12,000 psi. The eluent was collected on ice. The eluent was subjected to low speed centrifugation at 7500 g for 20 minutes, followed by high-speed centrifugation at 100,000 g for an hour. The supernatant from high-speed centrifugation was saturated with 20% ammonium sulfate by adding dry salt slowly with constant stirring. The sample was equilibrated for about 30 minutes and subjected to centrifugation at 16,000 g for 30 minutes. The pellet was discarded and the supernatant was adjusted to 60 % ammonium sulfate saturation by slowly  34  adding the remainder of the salt with continuous stirring. The sample was equilibrated for another 30 minutes at 4 °C and subjected to centrifugation at 16,000 g for 30 minutes at 4 °C. The supernatant was discarded. The precipitate was dissolved in a minimum volume of buffer A with PMSF (50uM) and dialyzed overnight against the same buffer. A n aliquot of the dialyzate was assayed for total protein and glucosidase activity. A fresh aliquot of PMSF (50 uM) was added to dialyzed sample, mixed well and applied at a flow rate of 0.5 ml/min to a Toyopearl D E A E column (2.5 x 20 cm) previously equilibrated with buffer A. Fractions were eluted with a step gradient of 0.1M NaCI, 0.3M NaCI and 0.5M NaCI in buffer A . Fractions eluted at a flow rate of 1.0 ml/min, were collected and assayed for protein and glucosidase I activity. The fraction with glucosidase I activity (0.3 M NaCI) was further purified with affinity chromatography using Concanavalin A Sepharose (Sigma C-7911) (1 x 2 cm ' column) as the affinity resin. Unbound protein was eluted with equilibration buffer (10 m M sodium phosphate, 0.5 M NaCI , 10 % glycerol, 0.1 m M MgCl, M n C l , 2  CaCl,, pH 6.8). The buffer was allowed to flow through the column by gravity. Glucosidase activity was eluted using Concanavalin A bumping buffer (20 m M sodium phosphate, 1.0 M a-methyl-D-glucoside, 10 m M EDTA. pH 6.8), dialyzed, and applied to a Mono Q F£R 5/5, anion exchange column attached to a FPLC system. Glucosidase activity was eluted using a continuous gradient of 0 - 1.0 M NaCI in buffer A (pH 6.8). The above experiments were conducted in duplicate.  35  3.5.2. Isolation of glucosidase from recombinant (pRANl) and native yeast (pHVX2) Crude cell extracts prepared from yeast cultures harbouring the shuttle vector p R A N l served as the source of recombinant glucosidase I. Crude cell extracts prepared from the yeast cultures harbouring the shuttle vector p H V X 2 served as the source of native glucosidase I (Control). The isolation procedure is summarized in Figure 3.  3.5.2.1. Growth of the culture A loopful of yeast cells was inoculated with 5 ml of the selective liquid medium in a small flask (Ausubel et al., 1994). The culture was allowed to grow overnight at 30 ° C with vigorous shaking at 300 rpm and mixed with 50 ml of fresh medium. The growth was continued. After 24 hours, the culture was transferred to a large flask with 195 ml fresh medium. The culture was allowed to grow for another 24 hours and 250 ml fresh medium was added to this growing culture. After 16 hours, the cultures were harvested. A total of two liters of culture broth was used for one extraction. The procedure was repeated with yeast transformants carrying plasmid pHVX2. This sample was used as the control.  3.5.2.2. Glass bead cell extraction and centrifugation Cells were harvested by centrifugation (7000 g for 5 minutes at 4 °C). The supernatant was discarded and the wet weight of the pellet was determined. The cells were washed with 10 ml of cold buffer A (10 m M sodium phosphate buffer, pH 6.8) and the centrifugation was repeated. The washed cells were re-suspended in the same buffer containing PMSF (50 ug /ml) as the protease inhibitor.  36  Overnight cultures of S. cerevisiae, minimal media + histidine (50ug/ml) (30°C; 300rpm)  I Centrifugation 7000g X 5 min 4° C  Yeast cell pellet + 3 X Buffer A (10 m M sodium phosphate, p H 6.8) + 4 X sterile acid washed glass beads, P M S F (50uM)  I Vortex (1 min) 4°C + store on ice 1 min ((Repeat 10 times)  Centrifugationat 16,000g x 20 min 4 °C  Pellet (discard)  Supernatant (Centrifugation at 100,000g; 60 min; 4°C)  Pellet (discard)  Supernatant (20-60% (NH4) S04, Precipitation; centrifugation 16,000g) 2  Supernatant (discard)  Pellet (Dissolve in 10 m M buffer A , Dialysis overnight against buffer A)  1 D E A E Sepharose Chromatography (Glucosidase activity eluted with 0.3M NaCl in 10 m M buffer A)  Dialysis overnight in 10 m M buffer A , ultrafiltration  I  F P L C Mono Q Glucosidase Activity eluted in 0.3 M NaCl in Buffer A) FPLC gel filtration with Superdex 200 HR 10/30 (Glucosidase activity eluted with retention volume of 12-13 mL 50 m M sodium phosphate  Figure 3. Soluble glucosidase I isolation scheme.  37  The volume of the buffer was about three times the volume of cells. A n aliquot of cell suspension (about 15 ml in each tube) and sterile glass beads (four times the wet weight of cells) were mixed in a 50 ml polypropylene centrifuge tube. Tightly closed tubes were mixed with a vortex mixer at highest speed for about one minute to break open the cells. Immediately after, the tube was cooled on ice for a period of one minute. This procedure of vortexing and cooling was repeated for 10 cycles and the tube was rested on ice. Once the mixture was separated, the liquid was poured into a centrifuge tube (50 ml) and stored on ice. The cell suspension with glass beads was vortexed one more time with 5 ml of buffer A with PMSF. The liquid portion was combined with the previously saved liquid fraction. This procedure was repeated until the protein was extracted completely from the remainder of the sample. Cell suspension was subjected to low speed centrifugation (16,000 g for 20 minutes at 4 ° C). The resultant supernatant was subjected to high-speed centrifugation (100,000 g for 60 minutes at 4 °C) and the supernatant from this step was used for ammonium sulfate precipitation.  3.5.2.3. Ammonium sulfate precipitation The supernatant from the high-speed centrifugation was subjected to ammonium sulfate precipitation (details in section 3.5.1).  3.5.2.4. DEAE-Sepharose anion-exchange chromatography The dialyzate from the previous section was applied at a flow rate of 0.5 ml/min to a D E A E Sepharose anion-exchange column (2.5 x 20 cm) (Pharmacia Co.) previously  38  equilibrated with buffer A (pH6.8) containing PMSF (50 uM). The sample was applied. Bio-Rad Econo System with a chart recorder was used to monitor the progress. The flow rate was adjusted to 1.0 ml/min. The column was washed with two column volumes of 10 m M buffer A (pH 6.8). After the protein peak was eluted, step gradients of the same buffer containing 0.1 M NaCI and 0.3 M NaCI were passed through the column. Protein peaks were collected and assayed for total protein and glucosidase activity. Eluents with glucosidase I activity were dialyzed overnight against buffer A (pH 6.8) and used for the next step.  3.5.2.5. Ultrafiltration The fraction with glucosidase activity was concentrated in an Amicon (Millipore, Bedford, M A ) stirred cell 8200 (10,000 MWCO), kept at 40 psi with nitrogen gas. The concentrated sample was assayed for total protein and glucosidase activity and stored at 4 °C.  3.5.2.6. FPLC with Mono Q  HR 5/5, anion exchange column  The concentrated sample (Section 3.5.2.5) was further purified using fast protein liquid chromatography (FPLC). Mono Q H R 5/5 (Pharmacia Co.), anion exchange column was used. Throughout the experiment flow rate was maintained at 0.2 ml/min. A l l buffers and samples were filtered through a 0.2 | i filter before loading. The column was equilibrated with two column volumes of buffer A (pH 6.8). The sample was eluted using a continuous buffer gradient of increasing salt concentration, ranging from 0.0-0.5 M NaCI in Buffer A. Protein peaks were collected and assayed for total protein. Pooled protein fractions were  39  assayed for glucosidase I activity, and further concentrated with Centricon Centrifugal Filter Device with YM-10 M W membranes (Millipore Corporation).  3.5.2.7. FPLC with Superdex 200 HR 10/30, gelfiltrationcolumn The enzyme from D E A E was further purified with gel filtration chromatography. FPLC grade Superdex 200 FIR10/30 gel filtration column (Amersham Pharmacia Biotech.) was used. Total bed volume was approximately 24 ml. All buffers were filtered and degassed. Before applying the sample, the column was equilibrated with two column volumes of 50 m M sodium phosphate (pH 6.8). A sample volume of 200 ul was injected using a syringe. The sample was eluted with the same buffer at a flow rate of 0.4 ml/min. Protein peaks were collected and assayed for total protein and glucosidase activity. Molecular weight of the fraction with glucosidase I activity was estimated using a plot (log molecular weight versus mobility), prepared with proteins of known molecular weights subjected to gel filtration chromatography, under the same conditions (Dhanawansa et al, 2002).  3.6. Enzyme assays 3.6.1. Glucosidase I activity assay During isolation, the enzyme activity of soluble fraction of the glucosidase I was monitored using a colorimetric assay which uses a synthetic trisaccharide a-D-Glc (1—> 2) aD-Glc (l->3) a-D-Glc-0 (CH ) g C O O C H as the substrate (Neverova et al, 1994). The 2  3  coupling enzyme assay for glucose detection (Fox and Robyt, 1991), modified for microplate reader, was used to assay the amount of glucose released from each fraction collected.  40  of synthetic trisaccharide were mixed in a microcentrifuge tube to initiate the reaction. The enzyme and the synthetic trisaccharide substrate were allowed to react for 15 minutes to one hour at 37 °C. The tubes were again briefly centrifuged to keep the reaction mixture together. The reaction was quenched using 25 ul of 1.25 M Tris-HCl (pH 7.6). Tris is a competitive inhibitor of glucosidase I. The reaction mixture was then transferred to a well on a microassay plate and 250 ul of developing solution containing glucose oxidase (5 units/ml), horseradish peroxidase (1 purpurogallin unit/ml) and o-dianisidine (3,3dimethoxybenzidine; Fast blue B) dihydrochloride (40 Ug/ml) in water was added. The solutions were protected from light by covering with aluminum foil and left to develop for 30 minutes at 37 ° C. Absorbance was read at 450 nm in a microplate reader (Lab Systems: E M S Reader MF). The amount of glucose released was calculated using the glucose standard curve. A standard glucose sample was also tested with each assay to check the reliability of the colour reagent. One unit of enzyme activity was defined as the amount of enzyme required to release one nmol of D-glucose, from the synthetic trisaccharide substrate, per minute at 37 ° C. The reaction scheme is summarized in Figure 4  3.6.2. A r y l glucosidase activity assay p-Nitrophenyl-oc-D-glucopyranoside (PNPG) assay was used to quantify aryl glucosidase activity. The assay conditions for the PNPG hydrolysis were as follows: 5 u,l of 20 m M PNPG and 15 jal of enzyme fraction in buffer A (pH 6.8) were incubated in a microcentrifuge tube at 37 ° C for 15 minutes to two hours. After centrifugation, the mixture was transferred to a well on the microassay plate. To this mixture, 300 ul of 200 m M  41  sodium carbonate was added and mixed to terminate the reaction. The absorbance was read at 405 nm. One unit of enzyme activity was defined as the amount of enzyme required for releasing one nmol of D-glucose from the PNPG substrate per minute, at 37°C. The extinction coefficient of 17,700 IVT'cm" was used for calculation (Neverova et al, 1994). 1  3 . 7 . Determination of the protein concentration The concentration of protein was determined using Bio-Rad Protein Assay. Manufacturer's instructions were followed. The protocol for assaying proteins with the BioRad dye reagent was based on the colorimetric determination of soluble proteins as described by Bradford (1976). Bovine serum albumin (BSA) was used as the standard. Duplicate samples of each protein concentration, ranging from 0.00 - 1.00 mg/ml B S A , were assayed. A standard curve was prepared and used to calculate the unknown protein concentration.  3 . 8 . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Samples were analyzed by SDS-PAGE (Laemmli, 1970) using Bio-Rad mini protein gel apparatus. The thickness of the gel was 1.5 mm. The stacking gels were 3% acrylamide and the separating gels were 11% acrylamide. The electrophoresis was performed at a constant current of 25 mA. Protein was visualized by staining with Coomassie brilliant blue R-250. The molecular weights of the fractions were determined by comparison with molecular weight standards (High Molecular Weight Range Sigma # M 3788).  42  Reducing Glucose 2(H 0), 0 2  +  aGlc ( 1 ^ 3 ) a G l c - 0 - R  2  •  Gluconic acid + H 0 2  o-Dianisidine  2  Peroxidase  (Colourless) T  Oxidized o-Dianisidine (Brown Colour)  Figure 4. Reaction pathway for glucosidase I activity assay. (Source: Neverova et al, 1994)  43  4.0. R E S U L T S A N D D I S C U S S I O N 4.1. Isolation of soluble glucosidase I from a yeast suspension prepared from "selfautolyzing" dry yeast cells Preliminary experiments were carried out for the isolation of the soluble fraction of the processing glucosidase I from yeast (Table 3). The cell suspension was prepared from a commercial dry yeast sample of S. cerevisiae purchased from Sigma Chemical Company. Dry yeast was chosen as the source of the enzyme since it has been used previously as the source of glucosidase I (Neverova et al., 1994; L i , 1999). The cell suspension was passed twice through a French Press at 12,000 psi to break cells. Successful use of the French Press to break open yeast cells were reported earlier (Neverova et al., 1996; L i , 1999). Preliminary trials were also carried out to check the suitability of the French press for breaking yeast cells. The crude cell homogenate collected on ice was subjected to low speed centrifugation (10,000 g) to remove cell debris. The supernatant collected from low speed centrifugation was subjected to high-speed centrifugation (100,000 g). The soluble fraction of the glucosidase I (supernatant) was separated from the membrane bound microsomal fraction (pellet). The soluble fraction of the enzyme was further purified using ammonium sulfate precipitation, Toyopearl D E A E anion exchange chromatography, Concanavalin A-Sepharose affinity chromatography and fast protein liquid chromatography with Mono Q FIR 5/5 anion exchange column. During isolation, the activity of the enzyme was monitored using a colorimetric assay, which uses a synthetic trisaccharide a-D-Glc (1—>2) a-D-Glc (1—>3) aD-Glc-0 (CH ) C O O C H as the substrate (Neverova et al., 1994). 2  8  3  44  Ammonium sulfate precipitation is the commonly used method for precipitating proteins from large volumes of crude preparations. It has been reported that 80% of any protein purification procedure use at least one step of precipitation with ammonium sulfate (Englard and Seifter, 1990). This ammonium sulfate precipitation was used in the present study because of its speed and large volume capability (Scopes, 1987). The procedure is also useful in concentrating the protein. Precipitation of proteins with ammonium sulfate deals with forces within and between polypeptide chains, as well as interactions of the protein molecules with water and other molecules in the solvating medium. In most proteins, interior part of protein molecule is shielded from the aqueous medium by hydrophobic side chains of amino acid residues. The surface of the protein interacts with the solvating medium through side chains of polar and ionic amino acid residues. Water molecules will hydrate the protein surface through formation of ion-dipole interactions and dipole interactions with the ionic and polar groups on side chains and polypeptide backbone (Li-Chan, 1996). Ammonium sulfate causes "salting out" of the protein in solution. This process involves neutralization of surface charges by ammonium sulfate and the reduction in the effective concentration of water. The concentration of ammonium sulfate required to cause "salting out" depends on the number and distribution of charged and polar groups on the surface of the protein, as well as the number and distribution of hydrophobic residues that are exposed and become dominant in intermolecular interactions as the charges are neutralized (Li-Chan, 1996). Using actively growing cultures of S. cerevisiae as the source of the enzyme, Kilker et al. (1981), determined the actual percentage of ammonium sulfate required to precipitate  45  glucosidase I. They reported that majority of the glucosidase I activity was concentrated in the 20-60% ammonium sulfate fraction. Previous studies have successfully adopted this procedure for the isolation of glucosidase I from dry yeast (Neverova et al, 1994; L i , 1999). The procedure was adopted in this study. Neverova et al. (1994) reported the successful use of anion exchange chromatography as a step for the isolation of glucosidase I. In their study, D E A E anion exchange chromatography and Mono Q H R 5/5 anion exchange chromatography were used. These procedures were adopted in this study. The salt gradient for Mono Q H R 5/5 elution was adjusted to 0.1-1.0 M NaCl. The salt gradient of 0.2-0.6 M NaCl was used in previous study. In ion exchange chromatography, separation of proteins is based on the reversible interaction between a charged protein and the oppositely charged chromatographic medium. Proteins bind to the column, as they are loaded. Conditions are then altered so that bound substances are eluted differentially. Since the bound molecules are subsequently displaced with the aid of an increasing salt gradient, proteins varying in charge can be separated During practical application of ion exchange chromatography, it is important to operate with pH values where the exchangers are mostly ionized and the protein contains an excess of positive or negative charges, e.g. they are not near their iso-electric point. The isoelectric point of glucosidase I was 4.8 (http://www.genedb.org). The p H optimum for glucosidase I was 6.8 (Kilker et al, 1981; Neverova et al, 1994; L i , 1999). D E A E is fully charged at this pH. Concanavalin A - Sepharose affinity chromatography step was introduced as an additional step for purification of glucosidase I. Concanavalin A-Sepharose affinity chromatography is used to partially purify glycoproteins that contain terminal mannose or  46  glucose residues (Ausubel et al.\ 1994). Separation is based on reversible interaction between sugars and the lectin, Concanavalin A. The terminal glucose residues of the target protein specifically and reversibly bind Concanavalin A. Unbound material and weakly bound proteins can be removed by washing the lectin with Concanavalin A running buffer. The bound protein can be eluted with a-methyl mannoside or a-mefhyl glucose. H P L C based mono Q HR 5/5 anion exchange chromatography step was introduced after Concanavalin A affinity chromatography step. Mono Q is a strong anion exchanger based on a beaded hydrophilic resin with one of the narrowest particle size (10 um). (Amersham - Pharmacia Biotech). The results of two replicate isolations are summarized in Table 3. Both isolations followed a similar trend. The increase in specific activity indicates that the protocol used was suitable for the isolation of glucosidase I. The 20-60% ammonium sulfate fraction had the highest enzyme activity (10-11 units/g). However, this enzyme activity may be an overestimation. The assay used to measure the activity of the enzyme was based on the absorbance of o-dianisidine (Figure 4). This crude enzyme fraction may have had interfering components causing high background absorbance. Neverova et al. (1994) stated that contaminating glucosidase II could act on disaccharide product of glucosidase I reaction increasing the amount of glucose detected. Another reason for higher reading may be due to the oxidation of the colour reagent odianisidine by endogeneous peroxidases. However, this was compensated for, by using appropriate blanks.  47  0.1 M NaCl  Flow through  0.3 M NaCl  0.5 M NaCl  c u  «  oo  © u c CQ I.  o vi <  0  100  20 Volume (ml)  Glucosidase I  Figure 5. DEAE Sepharose elution profile of ammonium sulfate precipitated fraction of processing a-glucosidase I from "self-autolyzing" dry yeast. The 20-60% ammonium sulfate fraction with glucosidase I activity was precipitated and dissolved in buffer A , dialyzed and then applied to D E A E column. The sample was applied at a flow rate of 0.5 ml/min. The flow rate was adjusted to 1.0 ml/min and the column was washed with two column volumes of 10 m M sodium phosphate (pH 6.8) buffer. The protein was eluted with step gradients of the same buffer containing 0.1 M , 0.3 M and 0.5 M NaCl.  48  Four protein peaks were collected and assayed after D E A E anion exchange chromatography (Figure 5). They were buffer A fraction, 0.1 M NaCl in buffer A fraction, 0.3 M NaCl in buffer A fraction and 0.5 M NaCl in buffer A fraction. Glucosidase I activity was concentrated in the protein fraction eluted with 0.3M NaCl in buffer A . The activity measurements from all other fractions eluted were insignificant. Proteins not bound to the column were eluted with buffer A . Lower glucosidase I activity reading in this fraction is an indication that the size of the column used was sufficient enough to bind all glucosidase I present in the sample. Step gradients of buffer A containing 0.1M NaCl and 0.5M NaCl removed other proteins bound to the column and eluted at different buffer strengths. The removal of glucosidase II and other aryl glucosidases from glucosidase I preparations was necessary as they may interfere with the activity assay of glucosidase I. Using p-nitro-phenyl cc-D-glucopyranoside (PNPG) assay, the activity of the aryl glucosidases were monitored. The results show that most of the column bound glucosidase II activity was present in the 0.1M NaCl fraction (Table 4). Kilker et al. (1981) also reported the removal of glucosidase II at a lower salt concentration during gradient elution from the D E A E Sephadex. Four-fold increase in the specific activity of glucosidase I (1 unit/mg to 4 units/mg) was achieved after D E A E anion exchange chromatography. The yield of the enzyme was reduced to 60-70 %. Concanavalin A-Sepharose affinity chromatography procedure was successful for further purification of glucosidase I. There was an increase in specific activity (13-23 fold in step III compared to 4-fold in step II. However, the yield of the enzyme was drastically  49  reduced (10 -17 % in step III compared to 62-71% in step II). The low yield may be the result of some contaminants interfering with the binding of the sample to the column. The amount of protein present was below the detection limit after the Mono Q FPLC step. This step was included to further purify glucosidase I to minimize the residual aryl glucosidases from the samples. Neverova et al. (1994) who developed this procedure for purification of glucosidase I reported that after Mono Q chromatography sample was free of contaminating glucosidase II. Overall, the isolation of the soluble fraction of the membrane bound glucosidase I from dry yeast was somewhat successful in isolating the enzyme. Specific activity data shows an increase in purity after each step. However, further characterization of the enzyme was not possible due to the insufficient yield of the isolated enzyme.  50  Vi Vi  Purification  Fold 03  *  2  S  -o ^ .2 t£  §'  g  £  <N  Specific Activity (Units/mg)  >>  -  —  ^  2  !  3  £  2  M  _  ^-  c>  ^  CN  g  1  tn  0  u 3  _ O  c o  _  H-*  c  "3 s  cu  a. Vi  3  CO _ Vi  CS  cu >->  Total Protein (mg)  a  „  •  _  -H  •  <o  O  C  Tt-  O  <=? 0\  <^  CN  O  >r»  «^  <=> O  1  1  " t -  0  <D DH  G  0 is  C  C  o  _  _ _  0.06  1.12  „  S '3  "e3  ^  Z  C  <=>  _  0  0  ^ 0  Step III  U  C  2 u OH PH  Step IV  CJ  Step I  O  (D  5  Step II  2  FPLC Mono Q Step IV  Vi  CM O  Step III  o  Purification Step  3  8 -o  O  £  6  cd  0  pq < Q  CN  «  -C ^  c o H—»  „  TJ  fcfl  a  __  o  t-> c  *&0> OH CO  "^j  I" H—*  t: ~  ' •T 3  £ —  5 3O  c o o c o 0  00 o c H-» -*-» S  W  <_  Replicate  "3D  O co  Concanavalin A  _3  •*  0.3 M NaCI DEAE  2 o u  7.77  GO  •c  Step II  08  J3 O  Cd  Step I  V  co <D T3  o  © -a  10.94  0.21  1.69  6.28  10.13 20-60% (NH4) SO 4  cu 'Ji  Total Activity (Units")  3  CO  Fraction  '5  •4—»  CN  51  2 DH  Table 4. Aryl glucosidase activity in fractions obtain from "self-autolyzing" yeast suspension  3  Fraction  Total Activity (Units )  Flow through D E A E  160  O.lMNaCl DEAE  140  0.3M NaCI D E A E  1.70  Concanavalin A  0.55  b  a  Results reported as per gram dry weight of yeast cells  b  One unit of enzyme activity was defined as the amount of enzyme required for releasing 1 nmol of D-glucose, from the PNPG substrate per minute, at 37°C.  52  4.2. Cloning, isolation and characterization of E. coli clones harbouring processing glucosidase I gene from S. cerevisiae The open reading frame (ORF) of the gene, CWH41 was PCR generated using S. cerevisiae genomic D N A as the template. Two primers were designed RD1 and RD2 (Table 2) to introduce two restriction sites (Bglll andXhol) at 5'end and 3'end for directional cloning. The PCR product (Appendix 1: Figure A) was digested overnight with BglWXhoI enzyme mixture and gel purified. The shuttle vector pHVX2 (Volschenk et al, 1997) with phosphoglycerate kinase (PGK1) promoter/terminator expression cassette and a synthetic polylinker EcoRl/BglWXhol  was used to subclone the PCR product. The vector pHVX2 was  also digested with BglWXhoI enzyme mixture and gel purified. The purified fragment containing ORF of CWH41was ligated with gel-purified fragment of pHVX2. The ligation mixture was incubated overnight at 16 °C, diluted and transformed with E. coli DH5oc. The positive clones were selected for the antibiotic resistance (Amp \ by growing them on L B + R  Ampicillin (100 (xg/ml) plates. Individual colonies were picked and plasmid D N A was isolated. Using restriction enzyme digestion and agarose gel electrophoresis, the presence of the insert was confirmed (Appendix I, Figure B). Two positive clones were isolated in the initial screening of about 120 colonies from the E. coli transformations. They were identified as clone #6 and clone #12. Yeast transformations were carried out using plasmid D N A isolated from the two clones of newly constructed plasmid (pRANl) and the control (plasmid pHVX2). The transformants were selected for LEU2 gene in host S. cerevisiae AH22.  53  EcoR I Bgl II Xho I  PGK1,  Bglll  PGK l  Xho  I  t  PCR generated insert  lacZ  Figure 6. The cloning strategy: CWH41 was cloned as a 2.5kb BglWXhoI into the BglWXhoI sites of p H V X 2 to yield p R A N l . (Source: Dhanawansa et al., 2002)  54  fragment  The vector and host systems were chosen because of several factors. First, the vector pHVX2 has the ampicillin resistance for the selection of E. coli and L E U 2 gene for the selection of yeast. It is also a multicopy plasmid. The high copy number of this plasmid is desirable to enhance the protein production. Plasmid pETVX2 has the constitutive promoter and terminator cassette with polylinker for expression of mRNA. Target genes can be subcloned to the polylinker region for them to be under the control of PGK1 promoter. Presence of terminator enhances the plasmid stability. Terminator containing plasmids usually prevent the read-through transcription from high activity promoters. Second, the E. coli host strain D H 5 a contains the recA mutation, which reduces recombination that may lead to instability of cloned D N A fragments (Yanish-Perron et al., 1985). Presence of E. coli endA mutation improves the quality of plasmid D N A . Third, S. cerevisiae host AH22 has recessive genes leu and his. Once grown in minimal media supplemented with L-histidine transformants with L E U 2 gene can be screened. Yeast cultures were prepared using these transformants grown at 30 °C in minimal media supplemented with L-histidine (Ausubel et al., 1994). Yeast cells from these transformants were observed under a phase contrast microscope. No difference in size or shape between the control and the recombinant yeast cells were noted. Both plasmids pHVX2 and p R A N l have PGK1 promoter and terminator. The difference is the presence of CWH41 region in the recombinant clones. Therefore, the enhancement in growth of the recombinant cultures must be the result of CWH41 gene under the control of PGK1 promoter. CWH41 gene has an indirect effect on cell wall assembly in S. cerevisiae (Parodi, 2000a). Cell wall assembly may have had an enhancing effect on cell growth. Mature cells can also reproduce mitotically. This can increase the number of cells  55  in the culture. Cultures with more cells reach the point of saturation faster. However, further studies are necessary to investigate the factors affecting the growth rate of recombinant clones in detail.  4.3. Purification of soluble glucosidase I isolated from S. cerevisiae strains harbouring the plasmid pRANl (recombinant) or plasmid p H V X 2 (control) The procedure used for the isolation of glucosidase I from recombinant and control yeast cultures was based on K i l k e r et al. (1981), and Neverova et al. (1994). Except for some modifications, the procedure used was very similar to the one used for the isolation of glucosidase I from dry yeast. Some improvements were made to the procedure used for dry yeast. Sterile acid washed glass beads were used to break yeast cells instead of the French Press. One reason for this change was to cut down the time between the cell breakage and centrifugation. When using the French Press, it was necessary to go to a different building to use the equipment, so there was a time period lapse between breaking the cells and the centrifugation. This may have contributed to loss of some enzyme activity. It is critical to speed up the early steps of isolation of the enzyme to minimize the damage to the enzyme by the action of proteases present in the crude homogenate. Glass bead extraction procedure and centrifugation can be carried out in the same tube. This is an additional advantage over the French Press method because it was less time consuming and reduced the possibility of contamination of the sample. A s pointed out earlier, dry yeast samples showed a significant drop in the yield of the enzyme after the Concanavalin A step (from 62-71% in D E A E to 17-10% in C o n A ) . The main objective at this point of the project was to establish that glucosidase I had been  56  overexpressed. Therefore, Concanavalin A-Sepharose isolation step was not included, as accurate estimates of activity could be determined after low-pressure anion exchange chromatography. The soluble fraction of the membrane bound processing glucosidase I was isolated using the cultures from yeast transformants from the recombinant clones #6 and #12 of p R A N l , and the control yeast (pHVX2). The results from yeast cultures harbouring plasmids with recombinant clones #6 and #12 of p R A N l grown in minimal media supplemented with L-histidine are presented in Table 5. The results from yeast cultures harbouring plasmid p H V X 2 grown in minimal media supplemented with L-histidine are presented in Table 6. The activity of glucosidase I isolated from the cultures harbouring the plasmid pHVX2 shows the basal level of activity of glucosidase I. The gene CWH41 encodes glucosidase I which is a membrane-bound enzyme. The soluble fraction of the membrane bound processing glucosidase I was isolated from overexpressed p R A N l . Only 10 % of the enzyme activity was associated with membrane bound fraction of p R A N l compared to 67 % in the control p H V X 2 (Dhanawansa et al, 2002). There was a proteolytic clipping of the membrane bound enzyme between residues Ala 24 and Thr 25 in the overexpressed enzyme liberating the soluble form of the enzyme (Dhanawansa et al, 2002).  57  Table 5. Purification of soluble glucosidase I isolated from recombinant S. cerevisiae (pRANl) clones #6 and #12 grown in minimal media supplemented with L-histidine with one hour assay time . 3  Sample  Purification Step  Fraction  Total Activity (Units")  Total Protein (mg)  Specific activity (Units/mg)  Yield (%)  Fold Purification  Step I  20-60%  162  18.4  9  100  1  Step II  0.3 M NaCI DEAE  93  1.4  66  57  7  Step I  20-60%  50  4.8  11  100  1  140  2.3  61  280  6  (NH4) S04 2  Clone #6  Clone #12  (NH4) S04 2  Step II  a b  0.3 M NaCI DEAE  Reported as per gram wet weight of yeast cell 1 Unit of activity = 1 nmol of glucose released from tri-saccharide substrate per min.  58  Table 6. Purification of soluble glucosidase I isolated from control S. cerevisiae (pHVX2) grown in minimal media supplemented with L-histidine with one hour assay time . 3  Replicate  Purification Step  Total Activity (Units)  Total Protein (mg)  Specific Activity (Units/mg)  Yield (%)  Fold Purification  20-60% (Nrl4)2S0  3.90  4.1  1  100  1  0.3 M NaCl DEAE  6.61  1.7  4  169  4  3.63  3.8  1  100  1  6.21  1.2  5  171  5  Fraction  b  Step I  4  #1 Step II  Step I  20-60% (NH4) S0 2  4  #2 Step II  a  b  0.3 M NaCl DEAE  Reported as per gram wet weight of yeast cell 1 Unit of activity = 1 nmol of glucose released from tri-saccharide substrate per min.  59  An increase in total protein concentration in recombinant clone #6 can be seen (18.4 mg/g in #6) compared to recombinant clone #12 (4.8 mg/g in #12) and control samples (4.1 mg/g and 3.8 mg/g). This may suggest that the cells from recombinant clone #6 were easier to break. Very noticeable increase in total activity in both clones (162 units/g in #6; 50 units/g in #12) can be seen from the isolations from both clones of p R A N l compared to the isolation from the culture of p H V X 2 (3.9 units/g and 3.6 units/g). Both plasmids p H V X 2 and p R A N l have the PGK1 promoter and terminator cassette. The difference is the location of CWH41 gene. In recombinant clones the expression of CWH41 gene was under the control of PGK1 promoter. In the yeast containing the plasmid pHVX2, CWH41 gene was under endogeneous control. Therefore, it is clear that the presence of PGK1 promoter itself is not responsible for over-expression of the gene to increase glucosidase I activity. The total enzyme activity in 20-60% ammonium sulfate fraction (step I) in one recombinant and both control samples were lower than that of D E A E fraction (step II). This may be due to an under-estimation of the glucosidase I activity in ammonium sulfate precipitation. This trend was not observed in the previous experiment with dry yeast homogenate. There is a possibility that some substances present in the fresh culture may have interfered with the assay. It has been reported that interfering substances such as ascorbic acid, catechol, cysteine, glutathione and acetylsalicylic acid can decrease the glucose value (Sigma diagnostic; glucose reagent Instruction Manual), which was used to calculate the glucosidase I activity. Alternatively, other unknown substances present in the 20-60% ammonium sulfate fraction of the fresh culture may have interfered with glucosidase I activity. Anion exchange chromatography may have removed these interfering substances.  60  The dry yeast sample may lack this substance or they may not be in the active state as it was dried and stored for some time. At the 20-60% ammonium sulfate step, the specific activity of the control sample (Table 6) and the dry yeast (Table 3) were about 1 unit/mg of protein. The recombinant samples show a specific activity of 9-11 units/mg of protein (Table 5). There is an increase in specific activity from 20-60% ammonium sulfate step (step I) to D E A E chromatography (step II). This increase was 4-fold in dry yeast (Table 3) and 4-5-fold in p H V X 2 (Table 6). This is an indication that both samples behave in a similar manner. Whether the yeast is in dry of wet form, the ratio of protein or the enzyme activity is similar. The increase in specific activity in recombinant samples was 6-7-fold (Table 5). This increase in specific activity was more than that of dry yeast (4-fold) or the control culture (4-5 fold). Overall, these results indicate that there is an overexpression of the CWH41 gene under the control of PGK1 system. Lilly et al. (2000) studied the expression of the alcohol acetyltransferase gene (ATF1) under the control of PGK1 promoter terminator system in S. cerevisiae. Their results show an increase in enzyme activity leading to the formation of of ethyl acetate (3- to 10fold), iso-amyl acetate (3.8 to 12-fold), and 2-phenylethyl acetate (2- to 10-fold). Their results also suggest the suitability PGK1 promoter terminator system for overexpression of an enzyme in yeast. The efficiency of expression of recombinant genes depends on several factors. Some of these factors are the copy number of the gene, the strength of the promoter on the expression vector, messenger R N A stability and the toxicity of the recombinant product. Although these factors were not investigated in this work, it is clear that this PGK1 promoter/  61  terminator system is suitable for the over-expression of the gene CWH41. No toxicity due to overexpression of the enzyme was noted. Using the recombinant clone #12, further purification of recombinant glucosidase I was achieved. Clone #12 was chosen on the basis of the yeast transformation. Transformants from clone #12 produce fewer but larger colonies. Therefore, more cells were available to start cultures. Many small colonies were produced by the transformants from clone #6. Another reason for selecting clone #12 was the loss of activity of the enzyme from clone #6. For some unknown reason, both clones #6 and #12 did not show any overexpression after re-transformation of yeast. Therefore, only basal expression of CWH41 was noted. Using E. coli strains carrying p R A N l , the enzyme activity was recovered only from clone #12. The results of the three separate replicate experiments for the purification of soluble glucosidase I isolated from recombinant yeast clone #12 are summarized in Table 7. The representative chromatography profiles for D E A E and F P L C Mono Q are shown in Figures 7 and 8 respectively. As expected all three replicates show a decrease in total protein and an increase in specific activity after each step. Replicate #3 showed a 43-fold purification. Replicate #1 and #2 showed 32-fold and 31-fold purification.  62  B  C  0.1 M NaCI elution  20  40  60  Volume (mL)  0.3 M NaCI elution  80  100  120  Glucosidase I  Figure 7. DEAE Sepharose elution profile of ammonium sulfate precipitated fraction of processing ct-glucosidase I from clone #12 of 5. cerevisiae pRANl. The 20-60% ammonium sulfate fraction with glucosidase I activity was precipitated and dissolved in buffer A, dialyzed and then applied to D E A E column. The sample was applied at a flow rate of 0.5 ml/min. The flow rate was adjusted to 1.0 ml/min and the column was washed with two column volumes of 10 m M sodium phosphate (pH 6.8) buffer. The protein was eluted with step gradients of the same buffer containing 0.1M and 0.3 M NaCI.  Note: The sensitivity setting of the U V detector was changed in this experiment. It was set at low sensitivity from A - B , high sensitivity from B-C.  63  80  0.035 'S  70  0.030  60  u> 0.025 >> « I.  I.  < E a o ®  0.010 0.005  05 e §»  40  0.015  orban*  00  50  0.020  30  \  J  I /  20 10  0.000  0  -0.005 0  10  20 Volume (ml)  30  40  Glucosidase I  Figure 8. FPLC anion exchange (Mono Q) chromatography of dialyzed, 0.3 M NaCl DEAE eluted fraction of the processing alpha glucosidase I from clone #12 of S. cerevisiae pRANl The sample was passed through the column at a flow rate of 1 ml/min. A stepwise gradient (10% Buffer B , 20%buffer B, 30% buffer B and 50% buffer B) was used to elute proteins. The processing glucosidase I activity occurred in the 0.3 M NaCl D E A E protein peak. The starting buffer (buffer A) was 10 mm sodium phosphate (pH 6.8). Buffer B was prepared by adding I M NaCl to buffer A .  64  »i  Table 7. Purification of soluble glucosidase I isolated from recombinant yeast clone #12 (pRANl) from 5. cerevisiae grown in minimal media supplemented with L-histidine with one hour assay time  3  Replicate  Purification Step  Fraction  Total Activity (Units ) 50  Total Protein (mg) 4.75  Specific Activity (Units/mg) 11  Yield (%)  Fold Purification  100  1  b  Step I  20-60% (NH4) S0 2  Step II  0.3 M NaCI DEAE  140  2.31  61  280  6  Step n i  0.3M NaCI FPLC Mono Q 20-60% (NFLOiSCu  14  .04  350  28  32  51  19.86  3  100  1  #1  Step I  #2  4  Step II  0.3 M NaCI DEAE  196  6.36  31  384  10  Step HI  0.3M NaCI FPLC Mono Q 20-60% (NH4) S04  19  .20  95  37  31  25  6.93  4  100  1  Step I  2  #3  a  b  Step II  0.3 M NaCI DEAE  34  3.14  11  136  3  Step HI  0.3M NaCI FPLC Mono Q  5  .04  125  20  31  Reported as per gram wet weight of yeast cell 1 Unit of activity = 1 nmol of glucose released from tri-saccharide substrate per min  65  As discussed earlier, glucosidase I activity was monitored using the synthetic trisaccharide. According to Neverova et al. (1994) an hour incubation of the enzyme and the substrate were necessary for assay of the activity of the enzyme. However, for the recombinant enzyme, a shorter incubation time was needed. Activity of the recombinant enzyme after an hour incubation of the substrate and the enzyme was an underestimation of the enzyme activity. When measuring any enzyme activity, the requirement is to convert less than 20% of the substrate to product, to keep the enzyme at approximately maximum velocity. The time required for 20% turn over of the substrate for the recombinant glucosidase was much less than an hour. With longer incubation period the substrate will be limited thus reducing enzyme activity. Therefore, the enzyme isolation procedure was repeated with the recombinant sample and the control. During this isolation, the glucosidase I activity was monitored using the same substrate. However, the assay time was reduced to 15 minutes. The results are summarized in Table 8. As seen in the table, the total activity difference between the control and the cloned sample were much larger. Compared to one-hour incubations (Table 7), total activity and the specific activity values show an increase. Therefore, the incubation time cloned samples should be reduced to 15 minutes.  66  Table 8: Purification of soluble glucosidase I isolated from recombinant yeast clone #12 (pRANl) or the control (pHVX2) from S. cerevisiae grown in minimal media supplemented with L-histidine with 15 minute assay time . a  Sample  Purification Step  Step I Control (pHVX2)  Step II  Total Activity (Units)  Total Protein (mg)  20  18.5  0.3 M NaCl DEAE  20  FPLC Mono Q  Specific Activity (Units/mg)  Yield (%)  Fold Purification  1  100  1  10.8  2  58  2  3.0  0. 3  10  15  10  20-60% (NH4) S04  459  18.2  25 .  100  1  0.3 M NaCl DEAE  408  3.6  113  89  5  FPLC Mono Q  95  0.2  567  21  23  Fraction  6  20-60% (NFLO2SO4  Step III  Recombinant yeast #12 (pRANl)  Step I  2  StepH  Step III  a  Reported as per gram wet weight of yeast cell 1 Unit of activity = 1 nmol of glucose released from trisaccharide substrate per min  67  The fractions with enzyme activity were subjected to SDS-PAGE under reducing conditions (Figure 9). As can be seen, eluent of Mono Q F P L C showed a major protein band at 98 kDa, representing glucosidase I (Lane 7). The significant feature of the purification is that the active peak from the Mono Q column is correlated with the appearance of a protein with a molecular weight of about 98,000 by SDS-PAGE. It is important to mention that the protein band in lane 7 of SDS-PAGE had a much higher specific activity than the specific activity reported in Table 8. The specific activity of the F P L C purified protein fraction was 567 units/mg. when synthetic trisaccharide was used as the substrate (Table 8). This was the reading for pooled fractions of FPLC. A single F P L C fraction with the highest specific activity (about 4000 units/mg) was used for SDS-PAGE.  68  c  Lanes:  s  c  s  c  s  1 2 3 4 5 6 7 8  Figure 9. SDS-Polyacrylamide gel electrophoretic analysis of fractions obtained during purification of processing a-glucosidase I from p H V X 2 (control or " C " ) and p R A N l (over-expressed sample or "S") Lane 1. Sigma molecular weight marker; Lanes 2 and 3: 20-60 % ammonium sulfate precipitated glucosidase I active fraction; Lanes 4 and 5:glucosidase I active fraction eluted from D E A E Sepharose chromatography; Lanes 6 and 7: glucosidase I active fraction eluted from Mono Q FPLC: Lane 8: Bio-Rad molecular weight marker. Lanes 2, 4 and 6 are from pHVX2 and lanes 3,5 and 7 are from p R A N l  69  4.4. Gel filtration chromatography Preliminary trials with FPLC gel filtration chromatography were successful. Gel filtration separates proteins with differences in molecular size as they pass through a column packed with a gel. Usually sample is added to the top of the gel bed. As eluent is added to the top, the migration of the molecules will start. Small molecules that diffuse into gel beads are delayed in their passage down the column. Large molecules cannot diffuse into the gel. Therefore, they leave the column first and smaller molecules come out last. The advantage of this method is that this technique can be used with a minimum sample volume. The buffer composition also does not directly affect the resolution of the sample. Therefore, this method can be used as the final step so the sample can be stored in the same buffer. The sample does not have to be dialyzed before application to the column. Therefore, the loss of activity due to dialysis can be avoided. Superdex 200 HR 10/30 column (Pharmacia Biotech.) was used. This column has a broad separation range of molecular weight of 10,000-600,000. The total bed volume of the column was about 24 ml. This column was designed for use with the F P L C System. The buffer used was 50 m M sodium phosphate (pH 6.8). A flow rate of 0.4 ml/min was maintained through the complete run. The elution profile is presented in Figure 10. The molecular weight of the glucosidase I was about 89 kDa. The results in Table 9 are in agreement with published data (Dhanawansa et al., 2002). Further studies are necessary for optimizing the conditions for a better resolution of the sample. The sample volume use was very small (200 u.1). Due to the dilution, the protein concentration of the sample was below detection limit. This problem can be corrected in the future by loading a sample with a higher concentration of protein.  70  c O  o  o UH  Ov  c o  C 3  OH  c  <u o  c o o o o  o CN  in  E  o o  S  ^  •a o  _»| OO  'O  oo <  vo  CJ  IT)  VH  p O  c '53 O VH  ca •a <u  £ /-N 00  *ca  00  d  w  in  in  vo  VO  o 3  VH  <U OH  c  8  <D 4-*  ca  o H  VH  •4—»  O0  -O 3 oo  .2  <L> T3  o s  c  in vo  oo  VO  CN CO  P-l < W  ca o o ca  u  o  o ca VH  CO  W  d Q  U ca Z CO  CN  ca  AE  u ca z  **-»  AE  c o  W  d Q  X  <u  -o PH  O  VH  <D CX 3 00  CL,  3  OH  CO  4-.  oo  o ca  ~ in °a V H  a  co  W < W  E  Q  o <£-  8 CO d « 3 bb oo•o > o  o  E  o  o «fci c C 5—3 "53 * c o E - o o CL, C H 2 "I M >> « _T •a c V- .t- c c ca «J VH  O  1  ca o  •c  o  - H  4^  C O  T3 <U T3  ca ca  *°  GO  CL, 3 00  c S o  oo •c 6  o o  X  ca <u  •c  VO C-  CN  CN  O  VH  CL, 00  a,  oo  CL,  <  PQ  00  s > CH •— oo O  o  c o  ca ca o  '>  *—•  c o o ca ca t- ~c '53 <0  O-.  O  g  E  OH •-)  03  Cj  O0  Pi  71  '  ~  c  « -2 O  H  3  o  72  As seen in Figure 10, glucosidase I active fraction was eluted with a broad peak. The activity was concentrated towards the middle part of the peak. This was a preliminary trial to see the possibility of using gel filtration as a step for further purification of the enzyme. Of the 265 units loaded on the column, 176 units (66%) were recovered after gel filtration. Therefore, both techniques (anion exchange and gel filtration chromatography) are suitable for purification of glucosidase I. Optimization of conditions may be necessary to increase the recovery.  73  •?  4.5. Stability of the processing glucosidase I at 4°C for a period of 48 days  Stability of the enzyme preparation ( F P L C M o n o Q fraction) in 0 . 3 M NaCI buffer A (pH 6.8) was studied over a period of 48 days at 4 °C. The original activity of the sample was 92 units. The results are summarized in the Figure 11.  120  Time (days)  12.  Figure 11. Stability of glucosidase I isolated from clone #12 of pRANl with FPLC (Mono Q) anion exchange chromatography over a period of 48 days  74  Initially there was a sharp decrease in enzyme activity. This may be due to some interaction between the enzyme and interfering substances present in the enzyme preparation. The enzyme preparation may have had proteases such as aspartyl, cysteinyl, or metallo- proteases that acted on the enzyme. However, a previous study had found that adding additional proteolytic inhibitors did not improve stability (Li, 1999). Some other compounds such as bovine serum albumin (BSA) and glycerol were also found to increase the stability ( L i , 1999). Using dry yeast samples, L i (1999) studied the stability of glucosidase I. Results are in agreement with the present study.  75  5.0. C O N C L U S I O N A N D R E C O M M E N D A T I O N S F O R F U T U R E R E S E A R C H In this research, the cloning, overexpression and partial purification of the soluble fraction of the processing glucosidase I from S. cerevisiae was accomplished. Directional cloning with P C R and over-expression of the gene C W H 4 1 , encoding processing glucosidase I in S. cerevisiae was successful. Approximately a 30-fold increase in total enzyme activity of the recombinant clones compared to the control, after M o n o Q anion exchange chromatography was noted. There were no adverse effects on the growth of the cultures and no morphological abnormalities were observed in the transformed yeast. Purification protocol based on ammonium sulfate precipitation, D E A E weak anion exchange chromatography, F P L C based M o n o Q strong anion exchange chromatography and F P L C based Superdex 200 H R 10/30gel filtration chromatography was established. Storage stability of the enzyme, prepared after F P L C M o n o Q, was studied. The results show that 80% of the activity was lost within two weeks. The enzyme was relatively stable after two weeks with only a gradual decrease from 20 %-10 %. Study of the naturally occurring enzyme was limited by the low abundance of the enzyme and the limited availability of the substrate. Over-expression of the gene encoding glucosidase I w i l l provide enough enzyme to study the structure and the mechanism of the enzyme. Research should be directed towards further purification and characterization of the soluble and membrane bound fractions of the enzyme. The promoter activity of P G K 1 is also increased by heat shock (Piper et al, 1988). This may have a positive effect on the expression gene C W H 4 1 . Future research is needed to explore the effect of heat shock on the promoter activity.  76  To avoid long procedures of isolation and to minimize the loss of enzyme activity by the proteases present in cells, extracellular expression of the gene encoding glucosidase I, should be considered. Studies are needed to better understand the catalytic mechanism of action of enzyme. The use of inhibitors to block the activity of the enzyme is already underway. Once the mechanism is understood, therapeutic drugs which work on the basis of blocking the glycoprotein process pathway by inhibiting glucosidase I can be developed. The availability of protease-deficient strains has been of great benefit to yeast biochemistry. Several protease deficient strains are available from the Yeast Genetic Stock Centre (Burgers, 1999).  77  6.0 REFERENCES Abeijon, C. and Hirschberg, C.B. (1992). Topography of glycosylation reactions in the endoplasmic reticulum. Trends Biochem. Sci., 17: 31-36.  Asano, N . 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U.S.A., 96: 11878-11882.  93  APPENDIX I Buffers used for agarose gel electrophoresis 10 X T B E (Per Liter) TrisBase Boric Acid 500 m M E D T A  2 X T B E Sample Buffer 108g 55g 40ml  TBE Glycerol Bromophenol Blue  2X 20% 0.2 mg/ml  Buffers used for denaturing SDS-PAGE 10 X Running Buffer  2 X SDS Sample Buffer  Tris (0.25M) Glycine (1.92 M ) SDS (1%)  Glycerol 2-Mercaptoethanol SDS Bromophenol blue Tris  20ml 10 ml 4.6 g 10 mg 1.5 g  Dissolve in about 75 ml dH^O. Adjust the pH to 6.8 with 12N HC1. Bring the final volume to 100 ml Store at 4 °C.  94  1  3  O R F of CWH41  Figure A. Agarose gel electrophoresis of PCR generated open reading frame (ORF) of the gene, CWH41.  The P C R product was digested overnight with BglWXhol enzyme mixture. The digest was electrophoresed on a 1.0 % agarose gel for 3 hours at a constant voltage of 4 0 m V . Lane 1, X EcoR lIHind III fragments (Marker). Lane 3, Bgl l\JXho\ digested P C R fragment of the open reading frame of C W H 4 1 .  95  |  20  2728  3132  ft  t  iH  Ml  Figure B. Test cuts of DNA from E. coli. clones of pRANl or p H V X 2 D N A samples were digested overnight with Bgl lVXho\ restriction enzyme mixture. Digests were electrophoresed on a 1.0 % agarose gel for 2 hours at a constant voltage of 40mV. Lanes 1 and 20, A. EcoR lIHind III fragments (Marker). Lane 18, uncut D N A from clone #6 of p R A N l . Lane 19, Bgl WXho\.digested D N A from clone #6 of p R A N l . Lane 27, uncut D N A from clone #12 of p R A N l . Lane 28, Bgl IVXhoL digested D N A from clone #12 of p R A N l . Lane 31, uncut D N A from pHVX2. Lane 32, Bgl WXhol digested D N A from pHVX2 (control). Arrows indicate the presence of insert with the cloned gene, CWH41.  96  5  4  3  1  I  I  2 1  1 I  Figure C . Restriction analysis of D N A from clone #12 of newly constructed shuttle vector p R A N l .  D N A samples were digested with Bglll, Xhol and Bgl WXhol mixture. Digests were electrophoresed on a 1.0 % agarose gel for 3 hours at a constant voltage of 40mV. Lane 1, A, EcoR lIHind III fragments (Marker). Lane 2, uncut D N A from clone #12 of p R A N l . Lane 3 Bgl II digested D N A from clone #12 of p R A N l . Lane 4, Xhol digested D N A from clone #12 of p R A N l . Lane 5, Bgl IVXhol digested D N A from clone #12 of pRANl.  97  A P P E N D I X II  Sequence for region of CWH41 1  A T G C T T A T T T CAAAATCTAA GATGTTTAAA A C A T T T T G G A  TACTAACCAG  51  CATAGTTCTC  CTGGCATCTG CCACCGTTGA TATTAGTAAA  CTACAAGAAT  101  TCGAAGAATA  TCAAAAGTTC A C G A A T G A A T  CTTTACTGTG  GGCACCGTAT  151  AGATCCAATT  GTTACTTTGG  TATGAGGCCC AGATATGTCC  ATGAAAGTCC  201  ACTAATTATG  GGTATCATGT  GGTTCAACAG TTTGAGTCAG  GATGGCTTAC  251  ATTCGTTAAG  ACATTTTGCA ACGCCTCAGG ATAAATTGCA  AAAGTATGGT  301  TGGGAAGTGT ATGATCCAAG AATTGGTGGT  351  AAAAAATAAC  TTGAACTTGA  CTGTTTATTT TGTAAAGAGC AAGAACGGGG  401  AAAATTGGTC  AGTGAGAGTT  CAAGGTGAGC CTTTGGATCC  CAAGAGACCA  451  TCTACAGCAT  CTGTCGTATT  GTACTTTAGT  CAAAATGGTG  GCGAGATAGA  501  TGGAAAATCT  TCCTTAGCAA TGATAGGTCA TGACGGCCCT  AATGACATGA  551  AATTCTTCGG ATATTCTAAA  GAATTAGGCG AGTATCATCT  TACAGTAAAG  601  GACAATTTTG  CAAAAATCCG GAATATGAAA  CCATGGAAGT  651  AGCACCAGGA AGTGACTGCT CTAAAACAAG TCATTTATCA  CTTCAAATCC  701  CGGATAAAGA AGTTTGGAAG  GCTCGTGATG TTTTCCAATC  TCTAGTTAGC  751  GATTC GATAC GTGATATACT  GGAAAAGGAA GAGACAAAGC AGCGTCCTGC  801  TGATTTAATA CCAAGTGTTT  TAACTATTAG  AAATTTGTAC  AATTTTAATC  851  CTGGTAATTT  TCATTATATA  CAAAAGACAT  TTGATTTGAC  CAAAAAAGAT  901  GGGTTCCAAT  TTGATATCAC  TTACAATAAA  CTTGGCACTA  CTCAAAGTAT  951  TTCCACCAGG GAACAAGTTA  CGGAGTTGAT TACTTGGTCA  CTAAATGAGA  1001  TAAACGCGCG TTTTGATAAG  CAGTTTAGTT  1051  ATTGAAAGCG TGGAGGTCAA AAGAAGATTT  GCTTTAGAGA  CGCTATCAAA  1101  CCTATTAGGA GGAATCGGTT  ATTTCTATGG  GAATCAACTA  ATTGATCGTG  1151  AAACAGAATT  TGATGAGAGC CAGTTTACAG  AGATCAAACT  GCTGAATGCA  1201  AAAGAGGAAG GTCCATTTGA  ACTGTTTACC  1251  TTTCCCACGT GGATTCTATT  GGGATGAAGG TTTCCATCTT  GTCACTACTT  98  AAAGAAGTTT  TTATTGATGA  TTGGAGAAGG TCCCGACTCA  AGCGTTCCGA GCCGTGGCTT CTACAAATTA  1301  TGGAGTATGA  TTTTGACCTT GCCTTTGAAA  TCTTAGCGAG  CTGGTTTGAA  1351  ATGATCGAAG  ATGATAGTGG TTGGATTGCT  AGAGAAATTA  TACTGGGTAA  1401  TGAGGCAAGG  AGTAAAGTTC CGCAGGAATT TCAGGTGCAA  AATCCCAATA  1451  TTGCTAATCC  GCCAACTTTA TTGCTAGCAT  TTAGTGAAAT  GCTTTCTAGG  1501  GCCATTGAAA  ACATCGGCGA TTTCAACAGT  GACAGCTACC  ACCAAGTCAT  1551  GTTCAATAGT  AGGACAGCCA AGTTTATGAC  GAACAATCTA  GAAGCCAATC  1601  CTGGCTTGCT  AACCGAATAT GCCAAGAAAA T T T A T C C T A A  GCTATTGAAG  1651  CACTATAATT  GGTTCAGAAA ATCTCAAACA GGACTTATTG  ATGAATATGA  1701  GGAAATATTG  GAAGATGAAG GAATATGGGA  TAAGATTCAT  AAGAACGAAG  1751  TTTATAGATG  GGTTGGGCGT ACCTTCACTC ATTGTTTGCC  AAGCGGTATG  1801  GATGACTATC  CTAGAGCACA ACCACCAGAT GTAGCAGAAT  TGAACGTAGA  1851  CGCATTAGCA  TGGGTGGGCG TTATGACAAG ATCCATGAAG  CAAATTGCTC  1901  ACGTGTTGAA  GTTAACACAG GACGAGCAAA GATATGCACA  AATTGAGCAA  1951  GAGGTGGTCG  AGAATCTGGA TTTGTTACAC  TGGAGTGAAA  ATGACAATTG  2001  CTACTGTGAT  ATTAGCATCG ATCCAGAAGA CGATGAGATT  AGGGAGTTTG  2051  TATGTCATGA  GGGTTACGTC TCCGTATTGC  CCTTTGCATT  GAAGCTAATC  2101  CCCAAAAACT  CACCCAAGCT AGAGAAAGTA GTTGCTTTGA  TGAGTGACCC  2151  AGAAAAAATC  TTTTCAGACT ACGGGCTGTT  ATCACTATCG  AGACAAGACG  2201  ACTATTTCGG  CAAGGATGAA AACTATTGGA  GAGGCCCAAT  TTGGATGAAT  2251  ATTAATTACT  TGTGTCTTGA CGCAATGAGA TACTACTACC  CAGAGGTGAT  2301  TCTCGACGTG  GCTGGTGAGG CTAGCAATGC CAAGAAACTG  TACCAAAGTT  2351  TAAAGATTAA  TCTCAGTAAC AACATATACA AAGTTTGGGA  AGAACAAGGT  2401  TATTGTTATG  AAAATTACAG TCCGATAGAT  GGTCATGGTA  CTGGTGCTGA  2451  GCATTTCACA  GGCTGGACAG CACTTGTTGT  CAACATCCTT  GGACGCTTCT  2501  GA  99  100  101  ~5  ?P  O < CL CD CO  CO  ^88? CM CM .. - - CD *- O c CM CM -JJ > > CO O O CL  Z Z CO  T3 CO  co  v:  LU j£  co m  —t £ o CO Q n Q.  Q  Q  tr Z  tr i9.Z  CL DC CL-J  CM JQ  N- CO CO  CO _  •g to cp CO  CQQ/  104  8>6> c\i JC  _ S  .  - CD  S 8 •§ > > « O  O  Q.  Z Z CO  lO  *LU  <6 CD o «5 S»H ir o  co Q  TJ Q.  CM  CM CM  ..  CM CM "JJ  > > « ,  CO TO  CO  9> CO co  1551  107  o '  8)1^"  CM  5c? 108  109  110  Ill  112  

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