Elements for improved expression of bovine somatotropin

• US 20090093023A1
• Filed: 01/17/2008
• Published: 04/09/2009
• Est. Priority Date: 01/19/2007
• Status: Abandoned Application

• Current Assignee: Eli Lilly and Company
• Original Assignee: Eli Lilly and Company
• Inventors: Frantz, Julia P., Smith, Hong Q., Bogosian, Gregg
• Application Number:
  US12/009,332
• Publication Number:
  US 20090093023A1
• Time in Patent Office: Days
• US Class Current: 435/69.4
• CPC Class Codes: C07K 14/655 Somatostatins
• CPC Class Codes: C12N 15/67 General methods for enhancing the expression

Abstract

The invention allows improved expression of heterologous polypeptides such as bovine somatotropin (bST). Novel compositions and methods are provided for production of bST from a native bST cDNA in transformed host cells such as E. coli. In particular, DNA segments comprising novel ribosome binding sites and adjacent sequences are provided, which in conjunction with a promoter sequence direct high level expression of bST from its native cDNA. The invention also provides expression constructs comprising the ribosome binding sites and methods for the use of such constructs.

Claims

1. A nucleic acid sequence comprising SEQ ID NO:

106 operably linked to a native bovine somatotropin (bST) cDNA sequence.

2. The nucleic acid sequence of claim 1, further defined as operably linked to a promoter.

3. A nucleic acid sequence comprising SEQ ID NO:

8 operably linked to a native bST cDNA sequence.

4. The nucleic acid sequence of claim 3, further defined as operably linked to a promoter.

23. A transformed host cell comprising the nucleic acid sequence of claim 4.

24. The host cell of claim 23, wherein the host cell is a prokaryotic cell.

25. The host cell of claim 24, wherein the host cell is an E. coli cell.

5. A recombinant construct comprising a ribosome binding site sequence operably linked to a native bST cDNA sequence, wherein

(i) the ribosome binding site sequence contains the subsequence DDAGGDD,

(ii) the central G of the subsequence is located 10 to 13 nucleotides 5′

of the ATG start codon of the cDNA sequence, and

(iii) the 6 to 9 nucleotides between the subsequence and the ATG start codon comprise at least four nucleotides that are adenine or thymine.

6. The recombinant construct of claim 5, further defined as operably linked to a promoter.

7. The recombinant construct of claim 6, wherein the promoter comprises the nucleic acid sequence of SEQ ID NO:

26.

8. The recombinant construct of claim 5, wherein the bST cDNA sequence encodes a polypeptide sequence selected from the group consisting of SEQ ID NO:

29, SEQ ID NO;

110, SEQ ID NO;

112 and SEQ ID NO;

113.

26. A transformed host cell comprising the recombinant construct of claim 6.

27. The host cell of claim 26, wherein the host cell is a prokaryotic cell.

28. The host cell of claim 27, wherein the host cell is an E. coli cell.

9. A recombinant construct comprising a ribosome binding site sequence at least 80% identical to SEQ ID NO:

106 and operably linked to a native bST cDNA sequence, wherein the last eight nucleotides at the 3′

-end of the ribosome binding site sequence comprise at least seven nucleotides that are adenine or thymine.

10. A recombinant construct comprising a ribosome binding site sequence at least 80% identical to SEQ ID NO:

106 and operably linked to a native bST cDNA sequence.

11. The recombinant construct of claim 10, further defined as operably linked to a promoter.

12. The recombinant construct of claim 11, wherein the promoter comprises the nucleic acid sequence of SEQ ID NO:

26.

13. The recombinant construct of claim 10, wherein the ribosome binding site sequence is at least 84% identical to SEQ ID NO:

106.

14. The recombinant construct of claim 10, wherein the ribosome binding site sequence is at least 88% identical to SEQ ID NO:

106.

15. The recombinant construct of claim 10, wherein the ribosome binding site sequence is at least 92% identical to SEQ ID NO:

106.

16. The recombinant construct of claim 10, wherein the ribosome binding site sequence is at least 96% identical to SEQ ID NO:

106.

17. The recombinant construct of claim 10, wherein the ribosome binding site sequence is selected from the group consisting of SEQ ID NOs:

1-7 and SEQ ID NOs;

9-25.

18. The recombinant construct of claim 10, wherein the ribosome binding site sequence comprises SEQ ID NO:

106.

19. The recombinant construct of claim 10, wherein the bST cDNA sequence encodes a polypeptide sequence selected from the group consisting of SEQ ID NO:

29, SEQ ID NO;

110, SEQ ID NO;

112 and SEQ ID NO;

113.

20. A transformed host cell comprising the recombinant construct of claim 11.

21. The host cell of claim 20, wherein the host cell is a prokaryotic cell.

22. The host cell of claim 21, wherein the host cell is an E. coli cell.

29. A method for producing bST in a transformed host cell, comprising:

(a) obtaining the host cell according to claim 20, 23 or 26; and

(b) culturing the host cell under conditions that induce gene expression from the cDNA sequence.