Cloning and expression of T5 DNA polymerase reduced in 3'- to-5' exonuclease activity

• US 5,270,179 A
• Filed: 01/28/1992
• Issued: 12/14/1993
• Est. Priority Date: 08/10/1989
• Status: Expired due to Term

• Current Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated), Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Chatterjee, Deb K.
• Primary Examiner(s): Low, Christopher S. F.
• Application Number:
  US07/826,945
• Time in Patent Office: 686 Days
• Field of Search: 435/69.1, 435/191, 435/193, 435/194, 435/172.3, 435/320.1, 435/252.3, 435/252.33, 435/69.7, 435/69.8, 536/27, 536/23.1, 536/23.2, 536/23.72, 536/24.1, 536/24.2, 536/23.4, 935/6, 935/14, 935/38, 935/72, 935/73
• US Class Current: 435/69.1
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Abstract

The present invention discloses a recombinant DNA molecule having a full length or a truncated T5 DNA polymerase structural gene, each encoding a processive, thioredoxin-independent DNA polymerase. The DNA polymerase of the invention may have 3'"'"'-to-5'"'"'exonuclease activity or may be substantially reduced in processive 3'"'"'-to-5'"'"'DNA exonuclease activity. A method for producing these enzymes is also disclosed, as is the proteins produced by this process. The present invention is also directed to a leader sequence important for expression of soluble T5 DNA polymerase protein.

Claims

1. In a method for producing a soluble protein comprising the steps of:

a) culturing a transformed E. coli cell containing a heterologous DNA molecule,

b) expressing the protein, and

c) isolating said protein from said host cell, the improvement which comprises using as the heterologous DNA molecule one comprising;

(1) a structural gene encoding a soluble protein having a leader sequence containing at least the amino acid sequence;

space="preserve" listing-type="equation">NH.sub.2 -Met-Lys-Ile-Ala-Val-Val-Asp-Lys-Ala-Leu; and

(2) a promoter heterologous to said structural gene, wherein said promoter and said structural gene are in such position and orientation with respect to each other that the structural gene for expression in said E. coli cell is under control of said promoter.

2. The method of claim 1, wherein the heterologous DNA molecule further comprises:

(3) an origin of replication which maintains the structural gene/promoter/origin of the replication combination in said E. coli cell.

3. The method of claim 1, wherein the leader sequence is the amino acid sequence:

space="preserve" listing-type="equation">NH.sub.2 -Met-Lys-Ile-Ala-Val-Val-Asp-Lys-Ala-Leu-

space="preserve" listing-type="equation">Asn-Asn-Thr-Arg-Tyr-Asp-Lys-His-Phe-Gln-

space="preserve" listing-type="equation">Leu-Tyr-Gly-Glu-Glu-Val-Asp-Val-Phe-His.

4. The method of claim 2, wherein the leader sequence is the amino acid sequence:

space="preserve" listing-type="equation">NH.sub.2 -Met-Lys-Ile-Ala-Val-Val-Asp-Lys-Ala-Leu-

space="preserve" listing-type="equation">Asn-Asn-Thr-Arg-Tyr-Asp-Lys-His-Phe-Gln-

space="preserve" listing-type="equation">Leu-Tyr-Gly-Glu-Glu-Val-Asp-Val-Phe-His.

5. The method of claim 1, wherein the promoter is inducible.

6. The method of claim 5, wherein the promoter is a lac promoter.

7. The method of claim 5, wherein the promoter is a tac promoter.

8. The method of claim 5, wherein the promoter is a lambda P_L promoter.

9. In a method for producing a protein comprising the steps of:

a) culturing a transformed E. coli cell containing a heterologous DNA molecule; and

b) isolating said protein from said host cell,

the improvement which comprises using as the heterologous DNA molecule one comprising;

(1) a structural gene encoding a protein having a T5 DNA polymerase activity with a substantially reduced 3'"'"'-to-5'"'"' exonuclease activity, and a leader sequence containing at least the amino acid sequence;

space="preserve" listing-type="equation">NH.sub.2 -Met-Lys-Ile-Ala-Val-Val-Asp-Lys-Ala-Leu, and

(2) a promoter heterologous to said structural gene, wherein said promoter and said structural gene are in such position and orientation with respect to each other that the structural gene for expression in said E. coli cell is under control of said promoter.

10. The method of claim 9, wherein the heterologous DNA molecule further comprises:

(3) an origin of replication which maintains the structural gene/promoter/origin of the replication combination in said E. coli cell.

11. The method of claim 9, wherein the leader sequence is the amino acid sequence:

space="preserve" listing-type="equation">NH.sub.2 -Met-Lys-Ile-Ala-Val-Val-Asp-Lys-Ala-Leu-

space="preserve" listing-type="equation">Asn-Asn-Thr-Arg-Tyr-Asp-Lys-His-Phe-Gln-

space="preserve" listing-type="equation">Leu-Tyr-Gly-Glu-Glu-Val-Asp-Val-Phe-His.

12. The method of claim 10, wherein the leader sequence is the amino acid sequence:

space="preserve" listing-type="equation">NH.sub.2 -Met-Lys-Ile-Ala-Val-Val-Asp-Lys-Ala-Leu-

space="preserve" listing-type="equation">Asn-Asn-Thr-Arg-Tyr-Asp-Lys-His-Phe-Gln-

space="preserve" listing-type="equation">Leu-Tyr-Gly-Glu-Glu-Val-Asp-Val-Phe-His.

13. The method of claim 9, wherein the promoter is inducible.

14. The method of claim 13, wherein the promoter is a lac promoter.

15. The method of claim 13, wherein the promoter is a tac promoter.

16. The method of claim 13, wherein the promoter is a lambda P_L promoter.

17. The method of claim 9, wherein the exonuclease activity is less than or equal to 1 unit/mg protein.

18. The method of claim 17, wherein the exonuclease activity is less than 0.1 unit/mg protein.

19. The method of claim 18, wherein the exonuclease activity is less than 0.003 unit/mg protein.

20. The method of claim 19, wherein the exonuclease activity is less than 0.0001 unit/mg protein.

21. In a method for producing a protein comprising the steps of:

a) culturing a transformed E. coli cell containing a heterologous DNA molecule and

b) isolating said protein from said host cell, the improvement which comprises using as the heterologous DNA molecule one comprising;

(1) a structural gene encoding a protein having a T5 DNA polymerase activity, an exonuclease activity of less than or equal to 1 unit/mg protein, and a leader sequence which is the amino acid sequence;

space="preserve" listing-type="equation">NH.sub.2 -Met-Lys-Ile-Ala-Val-Val-Asp-Lys-Ala-Leu-

space="preserve" listing-type="equation">Asn-Asn-Thr-Arg-Tyr-Asp-Lys-His-Phe-Gln-

space="preserve" listing-type="equation">Leu-Tyr-Gly-Glu-Glu-Val-Asp-Val-Phe-His;

(2) an inducible promoter selected from the group consisting of a lac promoter, a tac promoter, and a lambda P_L promoter which is heterologous to said structural gene, wherein said inducible promoter and said structural gene are in such position and orientation with respect to each other that the structural gene for expression in said E. coli cell is under control of said promoter; and

(3) an origin of replication which maintains the structural gene/promoter/origin of the replication combination in said E. coli cell.

22. The method of claim 21, wherein the exonuclease activity is less than 0.1 unit/mg protein.

23. The method of claim 22, wherein the exonuclease activity is less than 0.003 unit/mg protein.

24. The method of claim 23, wherein the exonuclease activity is less than 0.0001 unit/mg protein.

25. In a method for producing a soluble protein comprising the steps of:

a) culturing a transformed E. coli cell containing a heterologous DNA molecule;

b) expressing the protein; and

c) isolating said protein from said host cell,

the improvement which comprises using as the heterologous DNA molecule one comprising;

(1) a structural gene encoding a soluble T5 DNA polymerase having a leader sequence which is the amino acid sequence;

space="preserve" listing-type="equation">NH.sub.2 -Met-Lys-Ile-Ala-Val-Val-Asp-Lys-Ala-Leu-

space="preserve" listing-type="equation">Asn-Asn-Thr-Arg-Tyr-Asp-Lys-His-Phe-Gln-

space="preserve" listing-type="equation">Leu-Tyr-Gly-Glu-Glu-Val-Asp-Val-Phe-His;

(2) an inducible promoter selected from the group consisting of a lac promoter, a tac promoter, and a lambda P_L promoter heterologous to said structural gene, wherein said inducible promoter and said structural gene are in such position and orientation with respect to each other that the structural gene for expression in said E. coli cell is under control of said promoter, and

(3) an origin of replication which maintains the structural gene/promoter/origin of the replication combination in said E. coli cell.

26. In a method for producing a soluble protein comprising the steps of:

a) culturing a transformed E. coli cell containing a heterologous DNA molecule;

b) expressing the protein; and

c) isolating said protein from said host cell,

the improvement which comprises using as the heterologous DNA molecule one comprising;

(1) a structural gene encoding a protein having a T5 DNA polymerase activity, an exonuclease activity of less than or equal to 1 unit/mg protein, and a leader sequence which is the amino acid sequence;

space="preserve" listing-type="equation">NH.sub.2 -Met-Lys-Ile-Ala-Val-Val-Asp-Lys-Ala-Leu-

space="preserve" listing-type="equation">Asn-Asn-Thr-Arg-Tyr-Asp-Lys-His-Phe-Gln-

space="preserve" listing-type="equation">Leu-Tyr-Gly-Glu-Glu-Val-Asp-Val-Phe-His;

(2) an inducible promoter selected from the group consisting of a lac promoter, a tac promoter, and a lambda P_L promoter heterologous to said structural gene, wherein said inducible promoter and said structural gene are in such position and orientation with respect to each other that the structural gene for expression in said E. coli cell is under control of said promoter; and

(3) an origin of replication which maintains the structural gene/promoter/origin of the replication combination in said E. coli cell.

27. The method of claim 26, wherein the exonuclease activity is less than 0.1 unit/mg protein.

28. The method of claim 27, wherein the exonuclease activity is less than 0.003 unit/mg protein.

29. The method of claim 28, wherein the exonuclease activity is less than 0.0001 unit/mg protein.