Enhanced expression of human platelet-derived growth factor in Pichia pastoris

• US 6,194,169 B1
• Filed: 06/04/1998
• Issued: 02/27/2001
• Est. Priority Date: 06/04/1998
• Status: Expired due to Fees

• Current Assignee: University Of Kentucky Research Foundation (University of Kentucky)
• Original Assignee: University Of Kentucky Research Foundation (University of Kentucky)
• Inventors: Kaetzel, David M.
• Primary Examiner(s): Mertz, Prema
• Assistant Examiner(s): HAMUD, FOZIA M
• Application Number:
  US09/090,381
• Time in Patent Office: 999 Days
• Field of Search: 435/69.1, 435/69.4, 435/70.1, 435/71.1, 435/254.2, 435/254.23, 435/255.1, 435/255.5, 435/252.3, 435/69.8, 435/471, 435/320.1, 435/483, 435/325, 435/360, 435/366
• US Class Current: 435/69.1
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 14/49 Platelet-derived growth factor [PDGF]

Abstract

Enhanced yields of the platelet-derived growth factor (PDGF) B-chain are obtained using the Pichia pastoris yeast system. Yields of both wild-type and mutant human proteins are enhanced when the yeast is transformed with a vector containing the yeast mating factor promoter fused to a sequence encoding the mature protein. The secreted wild-type protein is indistinguishable from mature 29-32 kDa protein isolated from human and Chinese hamster ovary (CHO) cells. An RKK→EEE mutant exhibited reduced association with the cell surface and accumulated in the culture medium as 29-32 kDa forms. Stable transfection of U87 astrocytoma cells with RKK→EEE mutants of either the A- or B-chain inhibited malignant growth in athymic nude mice. Despite altered receptor binding activities, each mutant retained full mitogenic activity when applied to cultured Swiss 3T3 cells. Circular dichroism spectrophotometric analysis of the RKK→EEE mutant revealed a secondary structure indistinguishable from the wild type. Yields of PDGF protein are increased many fold and in less time over previous methods.

Claims

1. A method of producing a platelet-derived growth factor PDGF BB or mutant thereof comprising:

transforming Pichia pastoris yeast with an expression vector encoding the PDGF BB or mutant thereof fused to a secretory signal, wherein said PDGF BB is a PDGF BB homodimer and said mutant has mitogenic activity similar to that of said PDGF BB homodimer;

maintaining the transformed yeast under culture conditions effective to express the PDGF BB or mutant as the mature protein in the culture supernatant; and

isolating the protein from the supernatant.

2. The method of claim 1, wherein the PDGF is human in origin.

3. The method of claim 1, wherein the PDGF is wild-type.

4. The method of claim 1, wherein the PDGF is a mutant of wild-type.

5. The method of claim 1, wherein the PDGF BB homodimer is the human 29-32 homodimer.

6. The method of claim 1, wherein the secretory signal is the secretory signal of yeast α

mating factor.

7. The method of claim 1, wherein the expression vector is pPIC9 modified to contain a nucleotide sequence encoding PDGF.

14. The method according to claim 1, wherein said mutant is altered at the Arg159, Lys160 and Lys161 residues with an anionic or polar, uncharged amino acid.

16. The method according to claim 14, wherein said amino acid is glutamic acid or serine.

17. The method according to claim 16, wherein said mutant comprises the sequence

Glu159, Glu160, Glu161;

Ser159, Ser160, Ser161;

or

Ser159, Lys160, Lys161.

8. A culture of Pichia pastoris transformed with an expression vector encoding a PDGF BB protein or mutant thereof fused to a signal peptide, wherein said PDGF BB is a PDGF BB homodimer and said mutant has mitogenic activity similar to that of said PDGF BB homodimer.

9. The culture of claim 8 wherein the PDGF is human in origin.

10. The culture of claim 8, wherein the PDGF is wild-type.

11. The culture of claim 8, wherein the PDGF BB homodimer is the human 29-32 homodimer.

12. The culture of claim 8 wherein the signal peptide is the secretory signal of yeast α

mating factor.

13. The culture of claim 8, wherein the expression vector is pPIC9 modified to contain a nucleotide sequence encoding PDGF.

15. The culture according to claim 8, wherein said mutant is altered at the Arg159, Lys160 and Lys161 residues with an anionic or polar, uncharged amino acid.

18. The culture according to claim 15, wherein said amino acid is glutamic acid or serine.

19. The culture according to claim 18, wherein said mutant comprises the sequence

Glu159, Glu160, Glu161;

Ser159, Ser160, Ser161;

or

Ser159, Lys160, Lys161.