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A combinatorial genetic library approach to target heterologous glycosylation enzymes to the endoplasmic reticulum or the Golgi apparatus of Pichia pastoris
Author(s) -
Nett Juergen H.,
Stadheim Terrance A.,
Li Huijuan,
Bobrowicz Piotr,
Hamilton Stephen R.,
Davidson Robert C.,
Choi ByungKwon,
Mitchell Teresa,
Bobrowicz Beata,
Rittenhour Alissa,
Wildt Stefan,
Gerngross Tillman U.
Publication year - 2011
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/yea.1835
Subject(s) - pichia pastoris , glycosylation , golgi apparatus , biology , endoplasmic reticulum , glycoprotein , heterologous , secretory pathway , fusion protein , pichia , yeast , secretion , computational biology , biochemistry , n linked glycosylation , recombinant dna , microbiology and biotechnology , gene , glycan
To humanize the glycosylation pathway in the yeast Pichia pastoris , we developed several combinatorial genetic libraries and used them to properly localize active eukaryotic mannosidases and sugar transferases. Here we report the details of the fusion of up to 66 N ‐terminal targeting sequences of fungal type II membrane proteins to 33 catalytic domains of heterologous glycosylation enzymes. We show that while it is difficult to predict which leader/catalytic domain will result in the desired activity, analysis of the fusion protein libraries allows for the selection of the leader/catalytic domain combinations that function properly. This combinatorial approach, together with a high‐throughput screening protocol, has allowed us to humanize the yeast glycosylation pathway to secrete human glycoprotein with complex N ‐glycosylation. Copyright © 2011 John Wiley & Sons, Ltd.