Method for producing therapeutic proteins in
The present invention related to methods and compositions for producing therapeutic proteins in yeast cell lines, and in particular Pichia pastoris , lacking dipeptidyl aminopeptidase (DAP) activity. DAP activity has been eliminated by genetically modifying a Pichia pastoris cell line such that STE13 and DAP2 have been deleted.
1. A method for producing a recombinant protein having an X-P/A at the amino-terminus, wherein X is any amino acid and P/A is proline or alanine, in a Pichia host cell, wherein the recombinant protein is secreted into the host cell culture medium, in which STE13 and DAP2 has been deleted or disrupted, comprising:
a. transforming the Pichia cell with a polynucleotide vector encoding the protein having the X-P/A at the amino-terminus;
b. culturing the transformed Pichia cell in a medium under conditions which induce expression of the protein; and
c. isolating the protein from the culture medium.
2. The method of claim 1 where the Pichia cell is Pichia pastoris.
3. The method of claim 1 , wherein the host cell has been genetically engineered to produce glycoproteins comprising human-like N-glycans.
4. The method of claim 3 , wherein the host cell wherein the human-like N-glycans are selected from the group consisting of hybrid and complex N-glycans.
5. The method of claim 1 , wherein the host cell is genetically engineered to produce glycoproteins that have predominantly an N-glycan selected from Man 5 GlcNAc 2 , GlcNAcMan 5 GlcNAc 2 , GalGlcNAcMan 5 GlcNAc 2 , NANAGalGlcNAcMan 5 GlcNAc 2 , GlcNAcMan 3 GlcNAc 2 , GlcNAc (1-4) Man 3 GlcNAc 2 , Gal (1-4) GlcNAc (1-4) Man 3 GlcNAc 2 , and NANA (1-4) Gal (1-4) GlcNAc (1-4) Man 3 GlcNAc 2 .
6. The method of claim 1 , wherein the Pichia expresses a human glycoprotein.
7. A genetically modified Pichia cell in which the genomic DNA encoding DPPIII, STE13 and DAP2 has been deleted or disrupted from the Pichia cell genome.
8. The Pichia cell of claim 7 which is Pichia pastoris.
9. The method of claim 5 , wherein the host cell has been genetically engineered to produce glycoproteins comprising human-like N-glycans.
10. The method of claim 9 , wherein the human-like N-glycans are selected from the group consisting of hybrid and complex N-glycans.
11. The method of claim 5 , wherein the protein is a human glycoprotein.
12. The method of claim 1 wherein DPPIII, STE13 and DAP2 are deleted or disrupted in the host cell.