Cloning and expression of T4 DNA polymerase

• US 4,935,361 A
• Filed: 09/18/1986
• Issued: 06/19/1990
• Est. Priority Date: 09/18/1986
• Status: Expired due to Term

• Current Assignee: Yale University
• Original Assignee: Yale University
• Inventors: Lin, Tsung-Chung, Rush, John E., Konigsberg, William, Spicer, Eleanor
• Primary Examiner(s): NOT, DEFINED
• Application Number:
  US06/909,060
• Time in Patent Office: 1,370 Days
• Field of Search: 435/194, 435/172.3, 435/320, 435/68, 435/70, 435/253, 435/255, 435/849, 435/317.1, 435/252.3, 435/252.33, 935/29, 935/41, 935/56, 935/73
• US Class Current: 435/194
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Abstract

The DNA sequence encoding T4 DNA polymerase (gene 43 of T4 phase) has been successfully cloned, thereby providing a convenient source of this enzyme. The method involves the production of a gene 43 NH₂ -terminus fragment absent its promoter. The latter is ligated to a fragment containing the remaining COOH-terminus sequence but lacking base pairs beyond this terminus which heretofore caused destabilization effects in cloning. The full sequence of the DNA sequence has been elucidated. Corresponding cloning methods are disclosed.

Claims

1. A recombinant cloning vector comprising a DNA sequence encoding a polypeptide having bacteriophage T4 DNA polymerase activity.

2. A vector of claim 1, which is a plasmid.

3. A plasmid of claim 2, which is a replicable expression vector capable, in a transformant microorganism or cell culture, of expressing said DNA sequence.

4. A plasmid of claim 3, comprising an inducible promoter effective to control expression of said DNA sequence, whereby when said plasmid is used to transform a suitable host, said promoter can be turned off under conditions in which said host will grow and can then be turned on under corresponding inducing conditions.

5. A plasmid of claim 4, wherein said promoter is inducible by a temperature change or a repressor/inducer combination.

6. A plasmid of claim 5, wherein said inducible promoter comprises a combination in said plasmid of the phage PL promoter and temperature sensitive repressor gene CI857, or is IPTG inducible ptac promoter.

7. pTL43W, a plasmid of claim 5.

8. pTL43Q, a plasmid of claim 5.

12. A microorganism or cell culture transformed with the vector of claim 4.

13. A microorganism of claim 12 obtained by transforming an E. coli strain.

14. A method for producing a polypeptide having bacteriophage T4 DNA polymerase activity comprising expressing the T4 DNA polymerase-encoding DNA sequence of the vector of claim 1 in a recombinant host.

15. A method of claim 14, wherein said expression is controlled by an inducible promoter.

16. A method of claim 15, comprising growing said host under growth conditions whereunder said promoter is repressed and thereafter inducing said promoter to express said DNA sequences.

17. A method of claim 16, wherein the promoter is inducible by a temperature change or a repressor/inducer combination.

18. A method of claim 17, wherein the promoter comprises, in a plasmid containing said DNA sequence, a combination of the λ

phage PL promoter and the temperature sensitive repressor gene CI857, or said promoter is IPTG inducible ptac promoter where said host comprises a DNA sequence encoding the lac repressor.

19. A method of claim 18, wherein said vector is plasmid pTL43W.

20. A method of claim 18, wherein said vector is plasmid pTL43Q.

21. A method of producing a polypeptide having T4 DNA polymerase activity comprising:

transforming a host cell culture with a plasmid of claim 4 to obtain a recombinant host cell,

culturing said recombinant host cell under conditions permitting expression of the T4 DNA polymerase encoding sequence of said plasmid to produce T4 DNA polymerase, and

recovering said polypeptide in substantially pure form.

9. An isolated DNA molecule encoding a polypeptide having bacteriophage T4 DNA polymerase activity.

10. A DNA sequence of claim 9, ATGAAAGAA TTTTATATCT CTATTGAAAC AGTCGGAAAT AACATTGTTG AACGTTATAT TGATGAAAAT GGAAAGGAAC GTACCCGTGA AGTAGAATAT CTTCCAACTA TGTTTAGGCA TTGTAAGGAA GAGTCAAAAT ACAAAGACAT CTATGGTAAA AACTGCGCTC CTCAAAAATT TCCATCAATG AAAGATGCTC GAGATTGGAT GAAGCGAATG GAAGACATCG GTCTCGAAGC TCTCGGTATG AACGATTTTA AACTCGCTTA TATAAGTGAT ACATATGGTT CAGAAATTGT TTATGACCGA AAATTTGTTC GTGTAGCTAA CTGTGACATT GAGGTTACTG GTGATAAATT TCCTGACCCA ATGAAAGCAG AATATGAAAT TGATGCTATC ACTCATTACG ATTCAATTGA CGATCGTTTT TATGTTTTCG ACCTTTTGAA TTCAATGTAC GGTTCAGTAT CAAAATGGGA TGCAAAGTTA GCTGCTAAGC TTGACTGTGA AGGTGGTGAT GACGTTCCTC AAGAAATTCT TGACCGAGTA ATTTATATGC CATTCGATAA TGAGCGTGAT ATGCTATGG AATATATCAA TCTTTGGGAA CAGAAACGAC CTGCTATTTT TACTGGTTGG AATATTGAGG GGTTTGACGT TCCGTATATC ATGAATCGTG TTAAAATGAT TCTGGGTGAA CGTAGTATGA AACGTTTCTC TCCAATCGGT CGGGTAAAAT CTAAACTAAT TCAAAATATG TACGGTAGCA AAGAAATTTA TTCTATTGAT GGCGTATCTA TTCTTGATTA TTTAGATTTG TACAAGAAAT TCGCTTTTAC TAATTTGCCG TCATTCTCTT TGGAATCAGT TGCTCAACAT GAAACCAAAA AAGGTAAATT ACCATACGAC GGTCCTATTA ATAAACTTCG TGAGACTAAT CATCAACGAT ACATTAGTTA TAACATCATT GACGTAGAAT CAGTTCAAGC AATCGATAAA ATTCGTGGGT TTATCGATCT ACTTTTAAGT ATGTCTTATT ACGCTAAAAT GCCTTTTTCT GGTGTAATGA GTCCTATTA AACTTGGGAT GCTATTATTT TTAACTCATT GAAAGGTGAA CATAAGGTTA TTCCTCAACA AGGTTCGCAC GTTAAACAGA GTTTTCCGGG TGCATTTGTG TTTGAACCTA AACCAATTGC ACGTCGATAC ATTATGAGTT TTGACTTGAC GTCTCTGTAT CCGAGCATTA TTCGCCAGGT TAACATTAGT CCTGAAACTA TTCGTGGTCA GTTTAAAGTT CATCCAATTC ATGAATATAT CGCAGGAACA GCTCCTAAAC CGAGTGATGA ATATTCTTGT TCTCCGAATG GATGGATGTA TGATAAACAT CAAGAAGGTA TCATTCCAAA GGAAATCGCT AAAGTATTTT TCCAGCGTAA AGACTGGAAA AAGAAAATGT TCGCTGAAGA AATGAATGCC GAAGCTATTA AAAAGATTAT TATGAAAGGC GCAGGGTCTT GTTCAACTAA ACCAGAAGTT GAACGATATG TTAAGTTCAG TGATGATTTC TTAAATGAAC TATCGAATTA CACCGAATCT GTTCTCAATA GTCTGATTGA AGAATGTGAA AAAGCAGCTA CACTTGCTAA TACAAATCAG CTGAACCGTA AAATTCTCAT TAACAGTCTT TATGGTGCTC TTGGTAATAT TCATTTCCGT TACTATGATT TGCGAAATGC TACTGCTATC ACAATTTTCG GCCAAGTCGG TATTCAGTGG ATTGCTGCTA AAATTAATGA ATATCTGAAT AAAGTATGCG GAACTAATGA TGAAGATTTC ATTGCAGCAG GTGATACTGA TTCGGTATAT GTTTGCGTAG ATAAAGTTAT TGAAAAAGTT GGTCTTGACC GATTCAAAGA GCAGAACGAT TTGGTTGAAT TCATGAATCA GTTCGGTAAG AAAAAGATGG AACCTATGAT TGATGTTGCA TATCGTGAGT TATGTGATTA TATGAATAAC CGCGAGCATC TGATGCATAT GGACCGTGAA GCTATTTCTT GCCCTCCGCT TGGTTCAAAG GGCGTTGGTG GATTTTGGAA AGCGAAAAAG CGTTATGCTC TGAACGTTTA TGATATGGAA GATAAGCGAT TTGCTGAACC GCATCTAAAA ATCATGGGTA TGGAAACTCA GCAGAGTTCA ACACCAAAAG CAGTGCAAGA AGCTCTCGAA GAAAGTATTC GTCGTATTCT TCAGGAAGGT GAAGAGTCTG TCCAAGAATA CTACAAGAAC TTCGAGAAAG AATATCGTCA ACTGACTATA AGTATGCTGA GTAAAAACTG CGAACGATAT AGCGAATATG ATGATAAAGG TTGGCCAGGA TTTAAATGCC CGTTCCATAT TCGTGGTGTG CTAACTTATC GTCGAGCTGT TAGCGGTTTA GGTGTAGCTC CAATTTTGGA TGGAAATAAA GTAATGGTTC TTCCATTACG TGAAGGAAAT CCATTTGGTG ACAAGTGCAT TGCTTGGCCA TCGGGTACAG AACTTCCAAA AGAAATTCGT TCTGATGTGC TATCTTGGAT TGACCACTCA ACTTTGTTCC AAAAATCGTT TGTTAAACCG CTGGCGGGTA TGTGTGAATC GGCTGGCATG GACTATGAAG AAAAAGCTTC GTTAGACTTC CTGTTTGGCT GATAG.

11. A DNA sequence which is a mutation of that of claim 10 and which codes for a polypeptide having bacteriophage T4 DNA polymerase activity.

22. An isolated DNA sequence which hybridizes with a DNA sequence of claim 10, and encoding a polypeptide having bacteriophage T4 DNA polymerase activity.