Yeast strains producing mammalian-like complex n-glycans
AssigneeOxyrane UK Limited
InventorsSteven Christian Jozef Geysens, Wouter Vervecken
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Abstract
Described herein are methods and genetically engineered fungal cells useful for producing target molecules containing mammalian-like complex N-glycans or containing intermediates in a mammalian glycosyiation pathway.
Claims23
A method of producing a fungal cell capable of producing proteins comprising GlcNAcMan5GlcNAc2 N-glycans or GlcNAcMan3GlcNAc2 N-glycans, said method comprising:
- a) providing a fungal cell genetically engineered to produce proteins comprising Man5GlcNAc2 N-glycans or Man3GlcNAc2 N-glycans;
- b) introducing into said cell a nucleic acid encoding a GlcNAc-transferase I, wherein said nucleic acid comprises a nucleotide sequence encoding a targeting sequence to target the encoded GlcNAc-transferase I to an intracellular compartment, wherein expression of said GlcNAc-transferase I in said fungal cell produces proteins comprising GlcNAcMan5GlcNAc2 N-glycans or GlcNAcMan3GlcNAc2 N-glycans.
-48. (canceled)
An isolated fungal cell genetically engineered to produce proteins comprising:
- (a) GlcNAcMan3GlcNAc2 N-glycans, wherein said fungal cell is a first fungal cell that is genetically engineered to be deficient in OCH1 activity and comprises one or more nucleic acids encoding an α-1,2-mannosidase, a GlcNAc-transferase I, and a mannosidase II, wherein each of said one or more nucleic acids encoding said α-1,2-mannosidase, said GlcNAc-transferase I, and said mannosidase II comprises nucleotide sequences encoding targeting sequences to target each encoded protein to an intracellular compartment, wherein expression of said α-1,2-mannosidase, said GlcNAc-transferase I, and said mannosidase II in said first fungal cell produces proteins comprising GlcNAcMan3GlcNAc2N-glycans;
- or
- (b) GlcNAc2Man3GlcNAc2 N-glycans, wherein said fungal cell is a second fungal cell that is genetically engineered to be deficient in ALG3 activity and comprises one or more nucleic acids encoding a GlcNAc-transferase I and a GlcNAc-transferase II, wherein each of said one or more nucleic acids encoding said GlcNAc-transferase I and said GlcNAc-transferase II comprises nucleotide sequences encoding targeting sequences to target each encoded protein to an intracellular compartment, wherein expression of said GlcNAc-transferase I, and said GlcNAc-transferase II in said second fungal cell produces proteins comprising GlcNAc2Man3GlcNAc2 N-glycans.
-62. (canceled)
A substantially pure culture of Yarrowia lipolytica cells, a substantial number of which are genetically engineered to produce glycoproteins comprising Gal2GlcNAc2Man3GlcNAc2 N-glycans, wherein said cells are genetically engineered to be deficient in OCH1 activity and to comprise one or more nucleic acids encoding an α-1,2-mannosidase, a GlcNAc-transferase I, a mannosidase II, a GlcNAc-transferase II, and a galactosyltransferase, wherein each of said one or more nucleic acids encoding said α-1,2-mannosidase, said GlcNAc-transferase I, said mannosidase II, said GlcNAc-transferase II, and said galactosyltransferase comprises nucleotide sequences encoding targeting sequences to target each encoded protein to an intracellular compartment, wherein expression of said α-1,2-mannosidase, said GlcNAc-transferase I, said mannosidase II, said GlcNAc-transferase II, and a galactosyltransferase in said cells produces proteins comprising Gal2GlcNAc2Man3GlcNAc2 N-glycans.
-66. (canceled)
An isolated fungal cell genetically engineered to produce proteins comprising Gal2GlcNAc2Man3GlcNAc2 N-glycans, wherein said cell is genetically engineered to be deficient in OCH1 activity and to comprise one or more nucleic acids encoding an α-1,2-mannosidase, a GlcNAc-transferase I, a mannosidase II, a GlcNAc-transferase II, and a galactosyltransferase, wherein each of said one or more nucleic acids encoding said α-1,2-mannosidase, said GlcNAc-transferase I, said mannosidase II, said GlcNAc-transferase II, and said galactosyltransferase comprises nucleotide sequences encoding targeting sequences to target each encoded protein to an intracellular compartment, wherein expression of said α-1,2-mannosidase, said GlcNAc-transferase I, said mannosidase II, said GlcNAc-transferase II, and a galactosyltransferase in said cell produces proteins comprising Gal2GlcNAc2Man3GlcNAc2 N-glycans.
Dependencies are inferred from the claim text.