Glycosylation of molecules
Described herein are methods and genetically engineered cells useful for producing an altered N-glycosylation form of a target molecule. Also described are methods and molecules with altered N-glycosylation useful for treating a variety of disorders such as metabolic disorders.
1. A method of producing an altered N-glycosylation form of a target protein, the method comprising:
providing a Yarrowia lipolytica or an Arxula adeninivorans cell genetically engineered to express a protein capable of removing glucose residues from GlcMan 5 GlcNAc 2 or Glc 2 Man 5 GlcNac 2 and to comprise a deficiency in an Asparagine Linked Glycosylation 3 (ALG3) activity; and
introducing into the cell a nucleic acid encoding a target protein, wherein the cell produces the target protein in an altered N-glycosylation form relative to the N-glycosylation form of the target protein produced in a corresponding non-genetically engineered cell.
2. The method of claim 1 , further comprising isolating the altered N-glycosylation form of the target protein.
3. The method of claim 1 , wherein the target protein is an exogenous protein.
4. The method of claim 1 , wherein the target protein is an endogenous protein.
5. The method of claim 1 , wherein the target protein is a human protein.
6. The method of claim 1 , wherein the target protein is a pathogen protein, a growth factor, a cytokine, a chemokine, an antibody or antigen-binding fragment thereof, or a fusion protein.
7. The method of claim 6 , wherein said antigen-binding fragment is selected from the group consisting of Fab, F(ab′) 2 , Fv, and single chain Fv (scFv) fragments.
8. The method of claim 1 , wherein the altered N-glycosylation form comprises Man 5 GlcNAc 2 .
9. The method of claim 1 , wherein the altered glycosylation is Man 5 GlcNAc 2 .
10. The method of claim 1 , wherein the cell is further genetically engineered to be deficient in Outer Chain Elongation (OCH1)activity.
11. The method of claim 1 , wherein the cell is further genetically engineered to express a protein having glucosyltransferase activity.
12. The method of claim 11 , wherein the protein having glucosyltransferase activity is Asparagine Linked Glycosylation 6 (ALG6).
13. The method of claim 1 , wherein the cell is further genetically engineered to express an alpha-mannosidase.
14. The method of claim 13 , wherein the alpha-mannosidase is an alpha-1,2-mannosidase.
15. The method of claim 14 , wherein the alpha-1,2-mannosidase is mannosidase 1 (MNS1).
16. The method of claim 14 , wherein the alpha-1,2-mannosidase is targeted to the endoplasmic reticulum.
17. The method of claim 16 , wherein the alpha-1,2-mannosidase is a fusion protein comprising an alpha-1,2-mannosidase polypeptide and an HDEL endoplasmic reticulum retention peptide.
18. The method of claim 1 , wherein the protein capable of removing glucose residues from GlcMan 5 GlcNAc 2 or Glc 2 Man 5 GlcNAc 2 is a glucosidase II.
19. The method of claim 18 , wherein the cell is further genetically engineered to express ALG6 activity.
20. The method of claim 18 , wherein the cell is further genetically engineered to express ALG6 activity; and alpha-mannosidase activity.
21. The method of claim 18 , wherein the cell is further genetically engineered to be deficient in OCH1 activity; to express ALG6 activity; and to express alpha-mannosidase activity.
22. The method of claim 1 , wherein the protein capable of removing glucose residues from Man 5 GlcNAc 2 is a mutanase.
23. The method of claim 1 , wherein the protein capable of removing glucose residues from GlcMan 5 GlcNAc 2 or Glc 2 Man 5 GlcNAc 2 comprises one or both of the alpha and beta subunits of a glucosidase II.
24. The method of claim 1 , wherein the cell is not genetically engineered to be deficient in OCH1 activity.
25. The method of claim 1 , further comprising additional processing of the glycoprotein.
26. The method of claim 25 , wherein the additional processing comprises enzymatic or chemical treatment of the altered N-glycosylation form of the target protein.
27. The method of claim 1 , wherein the protein capable of removing glucose residues from GlcMan 5 GlcNAc 2 or Glc 2 Man 5 GlcNAc 2 comprises an endoplasmic reticulum retention peptide.
28. An isolated Yarrowia lipolytica or Arxula adeninivorans cell genetically engineered to express a protein capable of removing glucose residues from GlcMan 5 GlcNAc 2 or Glc 2 Man 5 GlcNAc 2 and to comprise a deficiency in an ALG3 activity.
29. The isolated cell of claim 28 , said cell being further genetically engineered to express a protein having glucosyltransferase activity.
30. The isolated cell of claim 28 , said cell further comprising a nucleic acid encoding a target protein, wherein the cell produces the target protein in an altered N-glycosylation form relative to the N-glycosylation form of the target protein produced in a corresponding non-genetically engineered cell.
31. The isolated cell of claim 28 , wherein the protein capable of removing glucose residues from GlcMan 5 GlcNAc 2 or Glc 2 Man 5 GlcNAc 2 comprises an endoplasmic reticulum retention peptide.
32. A method of producing an altered N-glycosylation form of a target protein, the method comprising contacting a target protein with a cell lysate prepared from a Yarrowia lipolytica or an Arxula adeninivorans cell genetically engineered to express a protein capable of removing glucose residues from GlcMan 5 GlcNAc 2 or Glc 2 Man 5 GlcNAc 2 and to comprise a deficiency in an ALG3 activity, wherein the contacting of the target protein with the cell lysate results in an altered N-glycosylation form of the target protein relative to the N-glycosylation form of the target protein produced from the cell lysate of a corresponding non-genetically engineered cell.
33. The method of claim 32 , said cell being further genetically engineered to express a protein having glucosyltransferase activity.
34. The method of claim 32 , wherein the protein capable of removing glucose residues from GlcMan 5 GlcNAc 2 or Glc 2 Man 5 GlcNAc 2 comprises an endoplasmic reticulum retention peptide.
35. A substantially pure culture of Yarrowia lipolytica or Arxula adeninivorans cells, wherein the culture comprises Yarrowia lipolytica or Arxula adeninivorans cells which are genetically engineered to express a protein capable of removing glucose residues from GlcMan 5 GlcNAc 2 or Glc 2 Man 5 GlcNAc 2 and to comprise a deficiency in an ALG3 activity.
36. The substantially pure culture of claim 35 , said genetically engineered cells being further genetically engineered to express a protein having glucosyltransferase activity.
37. The substantially pure culture of claim 36 , said genetically engineered cells further comprising a nucleic acid encoding a target protein, wherein the genetically engineered cells comprising a nucleic acid encoding a target protein produce the target protein in an altered N-glycosylation form relative to the N-glycosylation form of the target protein produced in a corresponding non-genetically engineered cell.
38. The substantially pure culture of claim 35 , wherein the protein capable of removing glucose residues from GlcMan 5 GlcNAc 2 or Glc 2 Man 5 GlcNAc 2 comprises an endoplasmic reticulum retention peptide.