Construction of a library of human glycosyltransferases immobilized in the cell wall of Saccharomyces cerevisiae.
Fifty-one human glycosyltransferases were expressed in Saccharomyces cerevisiae as immobilized enzymes and were assayed for enzymatic activities. The stem and catalytic regions of sialyl-, fucosyl-, galactosyl-, N-acetylgalactosaminyl-, and N-acetylglucosaminyltransferases were fused with yeast cell wall Pir proteins, which anchor glycosyltransferases at the yeast cell wall glucan. More than 75% of expressed recombinant glycosyltransferases retained their enzymatic activities in the yeast cell wall fraction and will be used as a human glycosyltransferase library. In increasing the enzymatic activities of immobilized glycosyltransferases, several approaches were found to be effective. Additional expression of yeast protein disulfide isomerase increased the expression levels and activities of polypeptide N-acetylgalactosaminyltransferases and other glycosyltransferases. PIR3 and/or PIR4 was more effective than PIR1 as a cell wall anchor when the Pir-glycosyltransferase fusions were expressed under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter. Oligosaccharides such as Lewis x, Lewis y, and H antigen were successfully synthesized using this immobilized glycosyltransferase library, indicating that the Pir-fused glycosyltransferases are useful for the production of various human oligosaccharides.
Shimma
Yoh-Ichi
Y
Research Center for Glycoscience, AIST, Tsukuba Central 6, Ibaraki 305-8566, Japan.
Saito
Fumie
F
Oosawa
Fumi
F
Jigami
Yoshifumi
Y
D001709:Biotechnology / Q000379:methods; D002473:Cell Wall / Q000201:enzymology* / Q000235:genetics; D003470:Culture Media; D004800:Enzymes, Immobilized; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D006023:Glycoproteins; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009844:Oligosaccharides / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
D003470:Culture Media; D004800:Enzymes, Immobilized; D005656:Fungal Proteins; D006023:Glycoproteins; D009844:Oligosaccharides; C423367:PIR1 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D016695:Glycosyltransferases