Expression of protein C

• US 4,959,318 A
• Filed: 10/29/1986
• Issued: 09/25/1990
• Est. Priority Date: 06/27/1985
• Status: Expired due to Term

• Current Assignee: Zymogenetics Incorporated (Bristol-Myers Squibb Company)
• Original Assignee: Zymogenetics Incorporated (Bristol-Myers Squibb Company)
• Inventors: Foster, Donald C., Murray, Mark J., Berkner, Kathleen L.
• Primary Examiner(s): Mays, Thomas D.
• Application Number:
  US06/924,462
• Time in Patent Office: 1,427 Days
• Field of Search: 435/68, 435/70, 435/91, 435/172.3, 435/235, 435/243, 435/317, 435/226, 435/247, 435/320, 435/317.1, 536/27, 935/11, 935/23, 935/32, 935/34, 935/57, 935/60, 935/70
• US Class Current: 435/226
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 9/6429 Thrombin (3.4.21.5)
• CPC Class Codes: C12N 9/6464 Protein C (3.4.21.69)
• CPC Class Codes: C12N 9/647 Blood coagulation factors not provided for in a preceding group or according to more than one of the proceeding groups
• CPC Class Codes: C12Y 304/21005 Thrombin (3.4.21.5)
• CPC Class Codes: C12Y 304/21069 Protein C activated (3.4.21.69)
• CPC Class Codes: Y10S 435/849 Escherichia coli

Abstract

Genomic and cDNA sequences coding for a protein having substantially the same biological activity as human protein C and recombinant transfer vectors comprising these sequences are disclosed.

Methods are disclosed for producing a protein which has substantially the same biological activity as human protein C. The protein, which may be in the form of activated protein C, is produced by mammalian host cells transfected with a plasmid capable of integration in mammalian host cell DNA. The plasmid includes a promoter followed downstream by a nucleotide sequence which encodes a protein having substantially the same structure and/or activity as human protein C, the nucleotide sequence being followed downstream by a polyadenylation signal.

Claims

1. A DNA sequence which codes for a protein having substantially the same biological activity as human protein C or human activated protein C, said sequence further coding for the pre-pro peptide of factor VII.

17. The DNA sequence of claims 1 or 2, wherein said protein comprises the light chain of protein C as shown in FIG. 2 from alanine, amino acid number 1, to leucine, amino acid number 155, and the heavy chain of human activated protein C as shown in FIG. 2 from leucine, amino acid number 170, to proline, amino acid number 419.

2. A DNA sequence which codes for a protein having substantially the same biological activity as human protein C or human activated protein C, said sequence further coding for the amino acid sequence Arg-Arg-Lys-Arg at the cleavage site between the light and heavy chains.

3. An expression vector capable of integration in mammalian host cell DNA, said expression vector including a promoter followed downstream by a nucleotide sequence which encodes a protein having substantially the same biological activity as human protein C or human activated protein C, said nucleotide sequence further encoding the pre-pro peptide of factor VII, said nucleotide sequence being followed downstream by a polyadenylation signal, wherein transcription of the nucleotide sequence is directed by the promoter.

18. The expression vector of claims 3 or 4, wherein said protein comprises the light chain of protein C as shown in FIG. 2 from alanine, amino acid number 1, to leucine, amino acid number 155, and the heavy chain of human activated protein C as shown in FIG. 2 from leucine, amino acid number 170, to proline, amino acid number 419.

4. An expression vector capable of integration in mammalian host cell DNA, said expression vector including a promoter followed downstream by a nucleotide sequence which encodes the pre-pro peptide of protein C operably linked to a sequence which encodes a protein having substantially the same biological activity as human protein C or human activated protein C, said nucleotide sequence further encoding the amino acid sequence Arg-Arg-Lys-Arg at the cleavage site between the light and heavy chains, said nucleotide sequence being followed downstream by a polyadenylation signal, wherein transcription of the nucleotide sequence is directed by the promoter.

5. Isolated mammalian cells transfected with an expression vector capable of integration in mammalian host cell DNA, said expression vector including a promoter followed downstream by a nucleotide sequence which encodes a protein having substantially the same biological activity as human protein C, said nucleotide sequence further encoding the pre-pro peptide of factor VII, said nucleotide sequence being followed downstream by a polyadenylation signal, wherein transcription of the nucleotide sequence is directed by the promoter.

19. The cells of claims 5 or 6, wherein said protein comprises the light chain of protein C as shown in FIG. 2 from alanine, amino acid number 1, to leucine, amino acid number 155, and the heavy chain of human activated protein C as shown in FIG. 2 from leucine, amino acid number 170, to proline, amino acid number 419.

6. Isolated mammalian cells transfected with an expression vector capable of integration in mammalian host cell DNA, said expression vector including a promoter followed downstream by a nucleotide sequence which encodes the pre-pro peptide of protein C operably liked to a sequence which encodes a protein having substantially the same biological activity as human protein C, said nucleotide sequence further encoding the amino acid sequence Arg-Arg-Lys-Arg at the cleavage site between the light and heavy chains, said nucleotide sequence being followed downstream by a polyadenylation signal, wherein transcription of the nucleotide sequence is directed by the promoter.

7. A method for producing a protein which, upon activation, has substantially the same biological activity as human activated protein C, comprising:

introducing into an isolated mammalian host cell an expression unit comprising a sequence which encodes a protein having substantially the same biological activity as human protein C, said sequence further coding for the prepro peptide of Factor VII;

growing said mammalian host cell in an appropriate medium;

isolating the protein product encoded by said expression unit and produced by said mammalian host cell; and

activating the protein product to produce a protein having substantially the same biological activity as human activated protein C.

8. The method of claim 7, further comprising introducing into the host cell, with said expression unit, a selectable marker.

9. The method of claim 7 wherein the activating step comprises cleavage of the protein product with a protease selected from the group consisting of α

-thrombin, trypsin, and Russell'"'"'s viper venom factor X activator.

10. The method of claim 7 wherein said nucleotide sequence further encodes the amino acid sequence Arg-Arg-Lys-Arg at the cleavage site between the light and heavy chains.

11. The method of claim 7 wherein said mammalian host cell is selected from the group consisting of COS, BHK and 293 cells.

12. A method for producing a protein which has substantially the same biological activity as human activated protein C, comprising:

introducing into an isolated mammalian host cell an expression unit comprising a sequence which encodes the pre-pro peptide of protein C operably linked to a sequence which encodes a protein having substantially the same biological activity as human activated protein C, said sequence further encoding the amino acid sequence Arg-Arg-Lys-Arg at the cleavage site between the light and heavy chains;

growing said mammalian host cell in an appropriate medium; and

isolating the protein product encoded by said expression unit and produced by said mammalian host cells.

13. The method of claim 12 wherein said mammalian host cell is selected from the group consisting of COS, BHK and 293 cells.

20. The method of any one of claims 7, 12, or 14, wherein said protein comprises the light chain of protein C as shown in FIG. 2 from alanine, amino acid number 1, to leucine, amino acid number 155, and the heavy chain of human activated protein C as shown in FIG. 2 from leucine, amino acid number 170, to proline, amino acid number 419.

14. A method for producing a protein which, upon activation, has substantially the same biological activity as human activated protein C, comprising:

introducing into an isolated mammalian host cell an expression unit comprising a sequence which encodes the pre-pro peptide of protein C operably linked to a sequence which encodes a protein having substantially the same biological activity as human protein C, said sequence further coding for the amino acid sequence Arg-Arg-Lys-Arg at the cleavage site between the light and heavy chains;

growing said mammalian host cell in an appropriate medium;

isolating the protein product encoded by said expression unit and produced by said mammalian host cell; and

activating the protein product to produce a protein having substantially the same biological activity as human activated protein C.

15. The method of claim 14 wherein the activating step comprises cleavage of the protein product with a protease selected from the group consisting of α

-thrombin, trypsin, and Russell'"'"'s viper venom factor X activator.

16. The method of claim 14 wherein said mammalian host cell is selected from the group consisting of COS, BHK and 293 cells.