Modification of protein glycosylation in methylotrophic yeast
View Patent ↗The present invention relates to methods and genetically engineered methylotrophic yeast strains for producing glycoproteins with mammalian-like glycosylation. The present invention also relates to vectors useful for generating methylotrophic yeast strains capable of producing glycoproteins with mammalian-like glycosylation. Glycoproteins produced from the genetically engineered methylotrophic yeast strains are also provided.
1. A genetically engineered Pichia strain which produces glycoproteins comprising a core N-glycan structure characterized by the formula, GalGlcNAcMan5 or fewer GlcNAc2, wherein said strain expresses (1) an α-1,2-mannosidase or an enzymatically active fragment thereof which converts Man8GlcNAc2 to Man5GlcNAc2, (2) an N-acetylglucosaminyltransferase I (or GnTI) or an enzymatically active fragment thereof which converts Man5GlcNAc2 to GlcNAcMan5GlcNAc2, and (3) a β-1,4-galactosyltransferase (GalT) or an enzymatically active fragment thereof, wherein the genomic OCH1 gene of said strain is inactivated, and wherein at least about 10% of GlcNAcMan5GlcNAc2 is converted to GalGlcNAcMan5GlcNAc2.
2. The strain of claim 1 , wherein said strain is a Pichia pastoris strain.
3. The strain of claim 1 , wherein said α-1,2-mannosidase or said fragment thereof is engineered to contain an ER-retention signal.
4. The strain of claim 3 , wherein said ER-retention signal comprises HDEL (SEQ ID NO: 1).
5. The strain of claim 1 , wherein said GnTI or said fragment thereof is of an origin of a species selected from the group consisting of rabbit, rat, human, plant, insect, nematode and protozoa.
6. The strain of claim 5 , wherein said GnTI or said fragment thereof is of a human origin.
7. The strain of claim 1 , wherein said GnTI or said fragment thereof is engineered to contain a Golgi-retention signal.
8. The strain of claim 7 , wherein said Golgi-retention signal comprises SEQ ID NO: 11.
9. The strain of claim 1 , wherein said GalT or said fragment thereof is of an origin of a species selected from the group consisting of rabbit, rat, human, plant, insect and nematode.
10. The strain of claim 9 , wherein said GalT or said fragment thereof is of a human origin.
11. The strain of claim 1 , wherein said GalT or said fragment thereof is engineered to contain a Golgi-retention signal.
12. The strain of claim 11 , wherein said Golgi-retention signal comprises SEQ ID NO: 11.
13. A method of recombinantly producing a glycoprotein, comprising obtaining a genetically engineered Pichia strain according to claim 1 , and expressing said glycoprotein from said strain.
14. The method of claim 13 , wherein said glycoprotein is selected from a protein of a bacterial, fungal, viral or mammalian origin.
15. The method of claim 13 , wherein said strain is a Pichia pastoris strain.
16. The method of claim 13 , wherein said α-1,2-mannosidase or said fragment thereof is engineered to contain an ER-retention signal.
17. The method of claim 16 , wherein said ER-retention signal comprises HDEL (SEQ ID NO: 1).
18. The method of claim 13 , wherein said GnTI or said fragment thereof is of an origin of a species selected from the group consisting of rabbit, rat, human, plant, insect, nematode and protozoa.
19. The method of claim 18 , wherein said GnTI or said fragment thereof is of a human origin.
20. The method of claim 13 , wherein said GnTI or said fragment thereof is engineered to contain a Golgi-retention signal.
21. The method of claim 20 , wherein said Golgi-retention signal comprises SEQ ID NO: 11.
22. The method of claim 13 , wherein said GalT or said fragment thereof is of an origin of a species selected from the group consisting of rabbit, rat, human, plant, insect and nematode.
23. The method of claim 22 , wherein said GalT or said fragment thereof is of a human origin.
24. The method of claim 13 , wherein said GalT or said fragment thereof is engineered to contain a Golgi-retention signal.
25. The method of claim 24 , wherein said Golgi-retention signal comprises SEQ ID NO: 11.
26. The strain of claim 1 , wherein said α-1,2-mannosidase is of an origin of a mammalian species or a fungal species.
27. The strain of claim 26 , wherein said fungal species is selected from Aspergillus or Trichoderma reesei .
28. The method of claim 13 , wherein said α-1,2-mannosidase is of an origin of a mammalian species or a fungal species.
29. The method of claim 27 , wherein said fungal species is selected from Aspergillus or Trichoderma reesei .