Isolation, characterization, and expression in Escherichia coli of the DNA polymerase gene from Thermus aquaticus.
The thermostable properties of the DNA polymerase activity from Thermus aquaticus (Taq) have contributed greatly to the yield, specificity, automation, and utility of the polymerase chain reaction method for amplifying DNA. We report the cloning and expression of Taq DNA polymerase in Escherichia coli. From a lambda gt11:Taq library we identified a Taq DNA fragment encoding an epitope of Taq DNA polymerase via antibody probing. The fusion protein from the lambda gt11:Taq candidate selected an antibody from an anti-Taq polymerase polyclonal antiserum which reacted with Taq polymerase on Western blots. We used the lambda gt11 clone to identify Taq polymerase clones from a lambda Ch35:Taq library. The complete Taq DNA polymerase gene has 2499 base pairs. From the predicted 832-amino acid sequence of the Taq DNA polymerase gene, Taq DNA polymerase has significant similarity to E. coli DNA polymerase I. We subcloned and expressed appropriate portions of the insert from a lambda Ch35 library candidate to yield thermostable, active, truncated, or full-length forms of the protein in E. coli under control of the lac promoter.
Lawyer
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Department of Microbial Genetics, Cetus Corporation, Emeryville, California 94608.
Stoffel
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Saiki
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Myambo
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Drummond
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Gelfand
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D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D003062:Codon; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli; D005786:Gene Expression Regulation; D015151:Immunoblotting; D008969:Molecular Sequence Data; D010957:Plasmids; D011993:Recombinant Fusion Proteins; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013824:Thermus / Q000201:enzymology*
D003062:Codon; D011993:Recombinant Fusion Proteins; D004259:DNA-Directed DNA Polymerase