title: A combinatorial genetic library approach to target heterologous glycosylation enzymes to the endoplasmic reticulum or the Golgi apparatus of Pichia pastoris.
abstract: 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.
authors.affiliation: GlycoFi Inc., 21 Lafayette Street, Suite 200, Lebanon, NH 03766, USA. Juergen_Nett@merck.com
authors.forename: Juergen H
authors.initials: JH
authors.lastname: Nett
authors.forename: Terrance A
authors.initials: TA
authors.lastname: Stadheim
authors.forename: Huijuan
authors.initials: H
authors.lastname: Li
authors.forename: Piotr
authors.initials: P
authors.lastname: Bobrowicz
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Hamilton
authors.forename: Robert C
authors.initials: RC
authors.lastname: Davidson
authors.forename: Byung-Kwon
authors.initials: BK
authors.lastname: Choi
authors.forename: Teresa
authors.initials: T
authors.lastname: Mitchell
authors.forename: Beata
authors.initials: B
authors.lastname: Bobrowicz
authors.forename: Alissa
authors.initials: A
authors.lastname: Rittenhour
authors.forename: Stefan
authors.initials: S
authors.lastname: Wildt
authors.forename: Tillman U
authors.initials: TU
authors.lastname: Gerngross
mesh_terms: D004721:Endoplasmic Reticulum / Q000201:enzymology*; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism*; D006056:Golgi Apparatus / Q000201:enzymology*; D008361:Mannosidases / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D015202:Protein Engineering; D021382:Protein Sorting Signals / Q000235:genetics; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D005964:Glucosyltransferases; D008361:Mannosidases