Methods for increasing N-glycan occupancy and reducing production of hybrid N-glycans in Pichia pastoris strains lacking Alg3 expression
AssigneeMerck Sharp & Dohme Corp.
InventorByung-Kwon Choi
Life of this patent
Estimating the term…
Abstract
Methods are disclosed for increasing the yield and N-glycosylation site occupancy of paucimannose or complex N-glycans of recombinant glycoproteins produced in a recombinant host cell lacking dolichyl-P-Man:Man5GlcNAc2-PP-dolichyl alpha-1,3 mannosyltransferase (Alg3p) activity. In particular, the present invention provides recombinant host cells for producing recombinant glycoproteins that comprise a disruption of the expression of an OS-9 family gene in the host cell and which overexpress one or more Trypanosoma brucei STT3 proteins.
Claims9
A host cell that does not display dolichyl —P—Man:Man5GlcNAc2-PP-dolichyl alpha-1,3 mannosyltransferase (alg3p) activity, Att1p activity, and osteosarcoma 9 (OS-9) protein activity comprising a nucleic acid molecule encoding a Trypanosoma brucei STT3 protein integrated into the genome of the host cell, wherein the host cell is a yeast or filamentous fungus and the OS-9 protein activity is Yos9p activity.
A host cell comprising:
- (a) a disruption in expression of the endogenous dolichyl-P-Man:Man5GlcNAc2-PP-dolichyl alpha-1,3 mannosyltransferase (ALG3) gene;
- (b) a disruption in the expression of endogenous osteosarcoma 9 (OS-9) family gene;
- (c) disruption in expression of an endogenous gene encoding ATT;
- and
- (d) a nucleic acid molecule encoding a Trypanosoma brucei STT3 protein integrated into the genome of the host cell, wherein the host cell is a yeast or filamentous fungus and the OS-9 family gene is a YOS9 gene.
A method for producing a heterologous glycoprotein, comprising;
- (a) providing a host cell that does not display dolichyl-P-Man:Man5GlcNAc2-PP-dolichyl alpha-1,3 mannosyltransferase (Alg3p) activity, osteosarcoma 9 (OS-9) protein activity, Att1p activity, and having integrated into the genome of the host cell a nucleic acid molecule encoding a Trypanosoma brucei STT3 protein and a nucleic acid molecule encoding the heterologous glycoprotein;
- and
- (b) culturing the host cell under conditions for expressing the heterologous glycoprotein to produce the heterologous glycoprotein, wherein the host cell is a yeast or filamentous fungus and the OS-9 family gene is a YOS9 gene.
Dependencies are inferred from the claim text.