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Characterization of a family B DNA polymerase from Thermococcus barophilus Ch5 and its application for long and accurate PCR.

Abstract：

:The family B DNA polymerase gene from the euryarchaeon Thermococcus barophilus Ch5 (Tba5) contains an open reading frame of 6198 base pairs that encodes 2065 amino acid residues. The gene is split by three inteins that must be spliced out to form the mature DNA polymerase. A Tba5 DNA polymerase gene without inteins (genetically intein-spliced) was expressed under the control of the pET-28b(+)T7lac promoter in E. coli Rosetta 2(DE3)pLysS cells. The molecular mass of the purified Tba5 DNA polymerase was about 90kDa consistent with the 90,470Da molecular mass calculated based on the 776 amino acid sequence. The optimal pH for Tba5 DNA polymerase activity was 7.5 and the optimal temperature was 70-75°C. The enzyme possessed 3'→5' exonuclease activity and was activated by magnesium ions. PCR amplification using Tba5 DNA polymerase enables high-yield for 1- to 6-kb target DNA products, while 8- to 10-kb target DNA products were amplified at low or inefficient levels. To simultaneously improve product yield and amplification fidelity, Tba5 plus DNA polymerase mixtures were constituted with various amounts of Tba5 DNA polymerase mixed with Taq DNA polymerase. The Tba5 plus DNA polymerase mixtures robustly amplified up to 25-kb λ DNA fragments. In addition, the PCR error rate of Tba5 plus3 and Tba5 plus4 mixtures were much lower than those of wild-type Tba5 DNA polymerase, Pfu DNA polymerase, Taq DNA polymerase, and Pfu plus DNA polymerase.

journal_name

Enzyme Microb Technol

journal_title

Enzyme and microbial technology

authors

Kwon KM,Kang SG,Sokolova TG,Cho SS,Kim YJ,Kim CH,Kwon ST

doi

10.1016/j.enzmictec.2016.02.004

subject

Has Abstract

pub_date

2016-05-01 00:00:00

pages

117-26

eissn

0141-0229

issn

1879-0909

pii

S0141-0229(16)30019-9

journal_volume

86

pub_type

杂志文章

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