Compounds, vectors and methods for expressing human, cytosolic phospholipase A.sub.2

• US 5,328,842 A
• Filed: 04/12/1993
• Issued: 07/12/1994
• Est. Priority Date: 04/17/1991
• Status: Expired due to Fees

• Current Assignee: Eli Lilly and Company
• Original Assignee: Eli Lilly and Company
• Inventors: White, Donald L., Chiou, Xue-Chiou C., Sharp, John D., Hoskins, JoAnn, Kramer, Ruth M.
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): PROUTY, REBECCA E
• Application Number:
  US08/046,508
• Time in Patent Office: 456 Days
• Field of Search: 435/69.1, 435/20.3, 435/71.2, 435/198, 435/172.3, 435/252.3, 435/252.33, 435/240.2, 435/320.1, 536/23.2, 935/14, 935/29, 935/32, 935/56, 935/70, 935/71, 935/73
• US Class Current: 435/352
• 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: C12N 15/85 for animal cells
• CPC Class Codes: C12N 9/18 Carboxylic ester hydrolases (3.1.1)

Abstract

The invention includes recombinant DNA compounds, vectors and methods useful for expressing an exceptionally rare, human, cytosolic phospholipase A₂ (cPLA₂) enzyme. The invention also includes a method for screening compounds to identify inhibitors of cPLA₂ which is believed to partake in several disease processes.

Claims

1. A gene consisting essentially of a recombinant DNA sequence that encodes a protein having the amino acid sequence of SEQ ID NO:

2.

2. The gene of claim 1 wherein said encoded protein has cytosolic phospholipase A₂ (cPLA₂) activity of greater than 6 micromoles per minute per milligram, an apparent molecular weight of approximately 100,000 daltons as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis, and is activated over the range of from 150 to 600 nM Ca⁺⁺.

3. The gene of claim 2 wherein said DNA sequence is the DNA sequence of SEQ ID NO:

1.

4. A recombinant DNA vector comprising a gene of claim 1.

5. A recombinant DNA vector comprising the gene of claim 3.

6. The vector of claim 5 selected from the group consisting of pECPLA22, pHDCPF, and pHDCPFS.

7. A recombinant host cell comprising the vector of claim 4.

8. The vector of claim 6 that is plasmid pECPLA22.

9. A recombinant host cell comprising the vector of claim 8.

10. The host cell of claim 9 that is E. coli K12×

E. coli B hybrid RR1/pECPLA22 which is on deposit with the NRRL under accession number 18775.

11. The vector of claim 6 wherein the vector is a eukaryotic expression vector selected from the group consisting of pHDCPF and pHDCPFS.

12. The vector of claim 11 that is plasmid pHDCPF.

13. A recombinant host cell comprising the vector of claim 11.

14. The host cell of claim 13 that is E. coli K12 DH5 alpha/pHDCPF and is on deposit with the NRRL under accession number 18772.

15. The vector of claim 11 that is plasmid pHDCPFS.

16. A recombinant host cell comprising the vector of claim 15.

17. The host cell of claim 16 that is E. coli K12 DH5 alpha/pHDCPFS and is on deposit with the NRRL under accession number 18773.

18. A host cell comprising a eukaryotic cell transformed with the vector of claim 4.

19. A host cell comprising a eukaryotic cell transformed with the vector of claim 5.

20. The host cell of claim 19 wherein the cell is the human embryonal kidney cell line 293 and the vector is pHDCPF or pHDCPFS.

21. The host cell of claim 19 wherein the cell is the hamster cell line AV12 and the vector is pHDCPF or pHDCPFS.