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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 Kung, Huijuan Li, Robert Miele, Juergen Nett, Stefan Wildt, Tillman Gerngross
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 Kung, Huijuan Li, Robert Miele, Juergen Nett, Stefan Wildt, Tillman Gerngross
Dartmouth Scholarship
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. …