Humanization of yeast to produce complex terminally sialylated glycoproteins.
Yeast is a widely used recombinant protein expression system. We expanded its utility by engineering the yeast Pichia pastoris to secrete human glycoproteins with fully complex terminally sialylated N-glycans. After the knockout of four genes to eliminate yeast-specific glycosylation, we introduced 14 heterologous genes, allowing us to replicate the sequential steps of human glycosylation. The reported cell lines produce complex glycoproteins with greater than 90% terminal sialylation. Finally, to demonstrate the utility of these yeast strains, functional recombinant erythropoietin was produced.
Hamilton
Stephen R
SR
GlycoFi Inc., 21 Lafayette Street, Suite 200, Lebanon, NH 03766, USA.
Davidson
Robert C
RC
Sethuraman
Natarajan
N
Nett
Juergen H
JH
Jiang
Youwei
Y
Rios
Sandra
S
Bobrowicz
Piotr
P
Stadheim
Terrance A
TA
Li
Huijuan
H
Choi
Byung-Kwon
BK
Hopkins
Daniel
D
Wischnewski
Harry
H
Roser
Jessica
J
Mitchell
Teresa
T
Strawbridge
Rendall R
RR
Hoopes
Jack
J
Wildt
Stefan
S
Gerngross
Tillman U
TU
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000378:metabolism; D004921:Erythropoietin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D006031:Glycosylation; D006801:Humans; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D015202:Protein Engineering; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012794:Sialic Acids / Q000378:metabolism; D012795:Sialoglycoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D014170:Transformation, Genetic
D011994:Recombinant Proteins; D012794:Sialic Acids; D012795:Sialoglycoproteins; D004921:Erythropoietin; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid