Gene for human epidermal growth factor and synthesis and expression thereof

• US 5,096,825 A
• Filed: 01/05/1987
• Issued: 03/17/1992
• Est. Priority Date: 01/12/1983
• Status: Expired due to Term

• Current Assignee: Chiron Corporation (Novartis Ag)
• Original Assignee: Chiron Corporation (Novartis Ag)
• Inventors: Barr, Philip J., Mullenbach, Guy T., Urdea, Mickey S., Valenzuela, Pablo, Merryweather, James P.
• Primary Examiner(s): Martinell, James
• Application Number:
  US07/004,212
• Time in Patent Office: 1,898 Days
• Field of Search: 435/91, 435/68, 435/172.3, 435/320, 435/253, 435/255, 435/69.1, 435/69.4, 435/252.33, 435/256, 435/320.1, 935/13, 935/28, 935/69, 935/73, 536/27
• US Class Current: 435/254.2
• CPC Class Codes: C07K 14/485 Epidermal growth factor [EGF], i.e. urogastrone

Abstract

A DNA having a base sequence coding for human epidermal growth factor has been synthesized in blocks, and cloned. Novel recombinant DNA transfer vectors containing said cloned DNA have been constructed. The codon usage of the sequence reflects the codon bias of yeast. The DNA sequence is useful for the large scale synthesis of human epidermal growth factor in yeast cells or microorganisms transformed by said recombinant DNA transfer vectors.

Claims

1. DNA of the sequence for human epidermal growth factor comprising ##STR6## encoding for physiologically active human epidermal growth factor, having a promoter-proximal end and a promoter-distal end, wherein the 5'"'"' terminal ATG designates the coding strand and the promoter-proximal end of the sequence.

2. DNA of the sequence for human epidermal growth factor comprising the sequence of claim 1 ligated at each end to double-stranded linkers containing recognition sites for restriction endonucleases.

3. DNA of the sequence for human epidermal growth factor according to claim 2 wherein both of said double-stranded linkers contain a recognition site for EcoRI restriction endonuclease.

4. DNA of the sequence for human epidermal growth factor according to claim 2 wherein both of said double-stranded linkers contain a recognition site for HgaI restriction endonuclease.

5. A DNA sequence of claim 1 further comprising DNA of a transfer vector.

6. A DNA expression vector which comprises a yeast promoter ligated at the promoter-proximal end of the sequence of claim 1, said sequence being under transcriptional control of said yeast promoter.

7. A DNA expression vector which comprises a yeast promoter ligated at the promoter-proximal end of the sequence of claim 1, said sequence being under transcriptional control of said yeast promoter;

and a terminator region ligated at the promoter-distal end of said sequence.

8. A DNA expression vector according to claim 7 wherein said terminator region is from yeast.

9. A DNA expression vector of claim 6 wherein said yeast promoter comprises the GAPDH yeast promoter.

10. A DNA expression vector of claim 6 wherein said yeast promoter comprises the ADH-1 promoter.

11. A DNA expression vector of claim 7 wherein said yeast promoter comprises the GAPDH yeast promoter and said terminator region comprises the ADH-1 terminator.

12. A DNA expression vector of claim 7 wherein said yeast promoter comprises the ADH-1 yeast promoter and said terminator region comprises the ADH-1 terminator.

13. A yeast transformed by the DNA expression vector of claim 6.

14. A yeast transformed by the DNA expression vector of claim 7.

15. A yeast transformed by the DNA expression vector of claim 8

16. A yeast transformed by the DNA expression vector of claim 9.

17. A yeast transformed by the DNA expression vector of claim 10.

18. A yeast transformed by the DNA expression vector of claim 11.

19. A yeast transformed by the DNA expression vector of claim 12.

20. The DNA expression vector plot 2 EGF-1.

21. The DNA expression vector pYEGF-1.

22. The DNA expression vector pYEGF-2.

23. The yeast GM3C2 transformed by pYEGF-1.

24. The yeast GM3C2 transformed by pYEGF-2.

25. E. coli D1210 transformed by plot 2 EGF-1.