UBC Theses and Dissertations
Cloning and over-expression of processing a-glucosidase I gene from Saccharomyces cerevisiae (CWH41) and purification of the soluble form of the enzyme Dhanawansa, Ranjani
Processing α-glucosidase I (E.C. 188.8.131.52) is a key enzyme in the N-linked (asparagine-linked) oligosaccharide process pathway. It cleaves the α -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 DNA 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 pRANl. 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. CWH41 was over expressed using S. cerevisiae host AH22. Yeast transformants of pRANl and pHVX2 (control), selected for LEU2 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, DEAE 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.
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