So it's a race between the tobacco farmers and the microbreweries to get into the antibody business...
Message 18886396
LEBANON, NEW HAMPSHIRE - Apr. 15, 2003 - GlycoFi, Inc. today announced the publication of a major scientific milestone in efforts to produce complex human glycoproteins in high capacity fungal production systems....
Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris
Byung-Kwon Choi*, Piotr Bobrowicz*, Robert C. Davidson*, Stephen R. Hamilton*, David H. Kung, Huijuan Li*, Robert G. Miele*, Juergen H. Nett*, Stefan Wildt*, and Tillman U. Gerngross
Thayer School of Engineering, Dartmouth College, Hanover, NH 03755
Communicated by Phillips W. Robbins, Boston Medical Center, Boston, MA, March 4, 2003 (received for review December 5, 2002)
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 -1,6-mannosyltransferase gene from P. pastoris, several combinatorial genetic libraries were constructed to localize active -1,2-mannosidase and human -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 -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 homogenous 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.
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* Present address: GlycoFi, Inc., 21 Lafayette Street, Suite 200, Lebanon, NH 03766.
To whom correspondence should be addressed at: Thayer School of Engineering, Dartmouth College, 8000 Cummings Hall, Hanover, NH 03755. E-mail: tillman.gerngross@dartmouth.edu.
The member who communicated this paper is a Science Advisor for GlycoFi, Inc.
www.pnas.org/cgi/doi/10.1073/pnas.0931263100 |
