Vectors with pectate lyase signal sequence

• US 5,576,195 A
• Filed: 12/09/1994
• Issued: 11/19/1996
• Est. Priority Date: 11/01/1985
• Status: Expired due to Term

• Current Assignee: Xoma Technology Ltd. (Xoma Corp.)
• Original Assignee: Xoma Corp.
• Inventors: Robinson, Randy R., Lei, Shau-Ping, Better, Marc, Wilcox, Gary L.
• Primary Examiner(s): MOSHER, MARY
• Application Number:
  US08/357,234
• Time in Patent Office: 711 Days
• Field of Search: 435/320.1, 435/252.3, 435/252.33, 435/69.1, 435/69.7, 435/69.8, 435/69.6, 536/23.4, 536/23.7
• US Class Current: 435/69.8
• 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 14/43 Sweetening agents, e.g. thaumatin, monellin
• CPC Class Codes: C07K 16/3046 Stomach, Intestines
• CPC Class Codes: C07K 16/462 Igs containing a variable region (Fv) from one specie and a constant region (Fc) from another
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/732 Antibody-dependent cellular cytotoxicity [ADCC]
• CPC Class Codes: C07K 2317/734 Complement-dependent cytotoxicity [CDC]
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C07K 2319/034 containing a motif for targeting to the periplasmic space of Gram negative bacteria as a soluble protein, i.e. signal sequence should be cleaved
• CPC Class Codes: C07K 2319/036 targeting to the medium outside of the cell, e.g. type III secretion
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 9/88 Lyases (4.)

Abstract

The invention relates to DNA coding for a pectate lyase signal peptide which has been inserted into plasmids to create secretion vectors which are capable of producing a chosen protein by directing transport across the bacterial membrane. The secretion vectors have been used to secrete extracellular thaumatin and extracellular antibody fragments. The proteins produced by these vectors have biological activity. The thaumatin is properly folded and the antibody fragments are capable of binding antigens on target cancer cells.

Claims

1. In a method for production of a protein in a gram-negative bacterial host which comprises transforming said host with a recombinant vector which contains a promoter sequence operably linked to a first DNA sequence encoding said protein, the improvement comprising employing a recombinant vector which comprises a second DNA sequence encoding a pectate lyase signal peptide sequence MetLysTyrLeuLeuProThrAlaAlaAlaGlyLeuLeuLeuLeuAlaAlaGlnProAlaMetAla, operably linked to said first DNA sequence encoding said protein, wherein said protein is foreign to said pectate lyase signal peptide.

2. The method of claim 1, wherein said host is E. coli.

3. The method of claim 1, wherein said vector is a plasmid.

4. The method of claim 1, wherein said protein is thaumatin.

5. The method of claim 1, wherein said protein is an antibody chain or a fragment of said chain that comprises the variable region.

6. The method of claim 1, wherein said promoter is an araBAD or lac promoter.

7. The method of claim 1, wherein the said second DNA sequence is ATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAA CCAGCGATGGCC.

8. An isolated DNA comprising:

DNA encoding a signal peptide for pectate lyase operably linked to DNA encoding a protein foreign to said pectate lyase, wherein said pectate lyase signal peptide has the sequence MetLysTyrLeuLeuProThrAlaAlaAlaGlyLeuLeuLeuLeuAlaAlaGlnProAlaMetAla.

9. The isolated DNA of claim 8, wherein the sequence of said DNA encoding a signal peptide is ATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAA CCAGCGATGGCC.

10. A recombinant vector comprising a DNA sequence encoding a pectate lyase signal peptide having the Sequence MetLysTyrLeuLeuProThrAlaAlaAlaGlyLeuLeuLeuLeuAlaAlaGlnproAlaMetAla, wherein said DNA encoding said pectate lyase signal peptide is operably linked to DNA encoding a protein foreign to said pectate lyase signal peptide.

11. The vector of claim 10, further comprising a DNA promoter sequence, said promoter DNA sequence being operably linked to said DNA sequence coding for said signal and protein sequences so as to permit expression of said protein.

12. The vector of claim 11, wherein said promoter is an araBAD or lac promoter.

13. The vector of claim 10, which is a plasmid.

14. The vector of claim 10, wherein said protein is thaumatin.

15. The vector of claim 10, wherein said protein is an antibody chain or a fragment of said chain that comprises the variable region.

16. A gram-negative bacterial host comprising the vector of claim 10.

17. The host of claim 16, wherein said vector is a plasmid.

18. The host of claim 16, wherein said protein is thaumatin.

19. The host of claim 16, wherein said protein is an antibody chain or fragment of said chain that comprises the variable region.

20. The host of claim 16, wherein said vector further comprises a DNA promoter sequence, said promoter DNA sequence being operably linked to said DNA sequence coding for said signal peptide and said protein sequence so as to permit expression of said protein in said host.

21. The host of claim 20, wherein said promoter is an araBAD or lac promoter.

22. The host of claim 16, wherein said host is E. coli.

23. The host of claim 22 which is strain MC1061 or JM103 transformed with said vector.

24. The vector of claim 10, wherein said DNA sequence is ATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAA CCAGCGATGGCC.

25. A gram-negative bacterial host comprising the vector of claim 24.

26. A method for production of a foreign protein in a bacterial host, which comprises culturing in a culture medium a gram negative host containing said vector of claim 10, and purifying said protein from the culture medium.

27. The method of claim 26, wherein said DNA sequence is ATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAA CCAGCGATGGCC.

28. A method for externalization of a protein from the cytoplasm of a gram-negative bacterial host, said method comprising:

(a) culturing a gram-negative bacterial host that has been transformed with the vector of claim 11 in a culture medium

(b) expressing said protein; and

.

(c) secreting said protein from said cytoplasm so as to externalize said protein out of said cytoplasm.

29. The method of claim 28, wherein said host is E. coli.

30. The method of claim 29, wherein said E. coli is strain MC1061 or transformed with said vector or JM103.

31. The method of claim 28, wherein said vector is a plasmid.

32. The method of claim 28, wherein said foreign protein is thaumatin.

33. The method of claim 28, wherein said protein is an antibody chain or a fragment of said chain that comprises the variable region.

34. The method of claim 28, wherein said promoter is an araBAD or lac promoter.

35. The method of claim 28, wherein said DNA sequence is ATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAA CCAGCGATGGCC.