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Proc Natl Acad Sci U S A. 2003 Apr 29;100(9):5022-7. Epub 2003 Apr 17.

Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris.

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1
Thayer School of Engineering, Dartmouth College, Hanover, NH 03755, USA.

Abstract

The secretory pathway of Pichia pastoris was genetically re-engineered to perform sequential glycosylation reactions that mimic early processing of N-glycans in humans and other higher mammals. After eliminating nonhuman glycosylation by deleting the initiating alpha-1,6-mannosyltransferase gene from P. pastoris, several combinatorial genetic libraries were constructed to localize active alpha-1,2-mannosidase and human beta-1,2-N-acetylglucosaminyltransferase I (GnTI) in the secretory pathway. First, >32 N-terminal leader sequences of fungal type II membrane proteins were cloned to generate a leader library. Two additional libraries encoding catalytic domains of alpha-1,2-mannosidases and GnTI from mammals, insects, amphibians, worms, and fungi were cloned to generate catalytic domain libraries. In-frame fusions of the respective leader and catalytic domain libraries resulted in several hundred chimeric fusions of fungal targeting domains and catalytic domains. Although the majority of strains transformed with the mannosidase/leader library displayed only modest in vivo [i.e., low levels of mannose (Man)(5)-(GlcNAc)(2)] activity, we were able to isolate several yeast strains that produce almost homogeneous N-glycans of the (Man)(5)-(GlcNAc)(2) type. Transformation of these strains with a UDP-GlcNAc transporter and screening of a GnTI leader fusion library allowed for the isolation of strains that produce GlcNAc-(Man)(5)-(GlcNAc)(2) in high yield. Recombinant expression of a human reporter protein in these engineered strains led to the formation of a glycoprotein with GlcNAc-(Man)(5)-(GlcNAc)(2) as the primary N-glycan. Here we report a yeast able to synthesize hybrid glycans in high yield and open the door for engineering yeast to perform complex human-like glycosylation.

PMID:
12702754
PMCID:
PMC154291
DOI:
10.1073/pnas.0931263100
[Indexed for MEDLINE]
Free PMC Article
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