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Title
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Isolation, characterization, and expression in Escherichia coli of the DNA polymerase gene from Thermus aquaticus.
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Authors
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F.C.Lawyer,
S.Stoffel,
R.K.Saiki,
K.Myambo,
R.Drummond,
D.H.Gelfand.
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Ref.
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J Biol Chem, 1989,
264,
6427-6437.
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PubMed id
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Abstract
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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.
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