title: Improved thermostability and PCR efficiency of Thermococcus celericrescens DNA polymerase via site-directed mutagenesis.
abstract: The Thermococcus celericrescens (Tcel) DNA polymerase gene, which contains a 2328-bp open reading frame that encodes 775 amino acid residues, was expressed in the Escherichia coli strain Rosetta(DE3)pLysS. The expressed enzyme was purified through heat treatment, HisTrap™ HP column chromatography and then HiTrap™ SP HP column chromatography. Tcel DNA polymerase has poor thermostability and PCR efficiency compared to those of other family B DNA polymerases. To improve thermostability and PCR efficiency, mutant Tcel DNA polymerases were created via site-directed mutagenesis. Specifically, we targeted the A752 residue for enhanced thermostability and the N213 residue for improved PCR efficiency. The mutant Tcel DNA polymerases all showed enhanced PCR efficiency and thermostability compared to those of the wild-type Tcel DNA polymerase. Specifically, the double mutant TcelA752K/N213D DNA polymerase had an approximately three-fold increase in thermostability over that of the wild-type enzyme and amplified a long 10-kb PCR product in an extension time of 2min. However, there was a small change in the 3'→5' exonuclease activity compared with that of the wild-type Tcel DNA polymerase, even though the mutation is in the ExoII motif. The double mutant TcelA752K/N213D DNA polymerase had a 2.6-fold lower error rate compared to that of Taq DNA polymerase. It seems that the double mutant TcelA752K/N213D DNA polymerase can be used in LA (long and accurate) PCR.
authors: Department of Genetic Engineering, Sungkyunkwan University, 300 Chunchun-dong, Jangan-gu, Suwon, Republic of Korea.
authors: Kee Pum
authors: KP
authors: Kim
authors: Sung Suk
authors: SS
authors: Cho
authors: Kang Keun
authors: KK
authors: Lee
authors: Man Hui
authors: MH
authors: Youn
authors: Suk-Tae
authors: ST
authors: Kwon
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA; D013696:Temperature; D019712:Thermococcus / Q000201:enzymology*
chemical_list: D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase