Cloning and expression of T4 DNA polymerase

Claim 2

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

Claim 3

A vector of claim 1, which is a plasmid.

Claim 4

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

Claim 5

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.

Claim 6

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

Claim 7

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.

Claim 8

pTL43W, a plasmid of claim 5.

Claim 9

pTL43Q, a plasmid of claim 5.

Claim 10

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

Claim 11

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

Claim 12

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.

Claim 13

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

Claim 14

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

Claim 15

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

Claim 16

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.

Claim 17

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

Claim 18

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.

Claim 19

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

Claim 20

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.

Claim 21

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

Claim 22

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

Claim 23

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.

Claim 24

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