title: 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.
authors.affiliation: Department of Biological Sciences, Sungkyunkwan University, 300 Chunchun-dong, Suwon, Republic of Korea.
authors.forename: Kyung-Min
authors.initials: KM
authors.lastname: Kwon
authors.affiliation: Korea Ocean Research & Development institute, PO Box29, Ansan 425-600, Republic of Korea.
authors.forename: Sung Gyun
authors.initials: SG
authors.lastname: Kang
authors.affiliation: Winogradsky Institute of Microbiology, Russian Academy of Sciences, Prospect 60-Letiya Oktyabrya 7/2, 117312 Moscow, Russia.
authors.forename: Tatyana G
authors.initials: TG
authors.lastname: Sokolova
authors.affiliation: Department of Biotechnology and Bioengineering, Sungkyunkwan University, 300 Chunchun-dong, Suwon, Republic of Korea.
authors.forename: Sung Suk
authors.initials: SS
authors.lastname: Cho
authors.affiliation: Korea Ocean Research & Development institute, PO Box29, Ansan 425-600, Republic of Korea.
authors.forename: Yun Jae
authors.initials: YJ
authors.lastname: Kim
authors.affiliation: Department of Biological Sciences, Sungkyunkwan University, 300 Chunchun-dong, Suwon, Republic of Korea.
authors.forename: Cheorl-Ho
authors.initials: CH
authors.lastname: Kim
authors.affiliation: Department of Biotechnology and Bioengineering, Sungkyunkwan University, 300 Chunchun-dong, Suwon, Republic of Korea. Electronic address: stkwon@skku.ac.kr.
authors.forename: Suk-Tae
authors.initials: ST
authors.lastname: Kwon
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019766:Genes, Archaeal; D016133:Polymerase Chain Reaction / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics
chemical_list: D019843:Archaeal Proteins; D011994:Recombinant Proteins; D004259:DNA-Directed DNA Polymerase
keywords: Fidelity; Long and accurate PCR (LA PCR); PCR amplification; Thermococcus barophilus Ch5 DNA polymerase (Tba5)