This Article Full Text Full Text (PDF) Other Versions of this Article: AEM.01378-06v1 72/11/7003    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Shimma, Y.-i. Articles by Jigami, Y. Search for Related Content PubMed PubMed Citation Articles by Shimma, Y.-i. Articles by Jigami, Y. Agricola Articles by Shimma, Y.-i. Articles by Jigami, Y.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, November 2006, p. 7003-7012, Vol. 72, No. 11
0099-2240/06/$08.00+0     doi:10.1128/AEM.01378-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Construction of a Library of Human Glycosyltransferases Immobilized in the Cell Wall of Saccharomyces cerevisiae

Research Center for Glycoscience, AIST, Tsukuba Central 6, Ibaraki 305-8566, Japan

Received 15 June 2006/ Accepted 19 August 2006

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.

Published ahead of print on 25 August 2006.