Cloning and overexpression of thermostable DNA polymerase gene in Escherichia coli.
Thermostable DNA polymerase genes had been amplified from Thermus aquaticus YT-1 using the PCR technique. The amplified 2.5-kb DNA fragment was inserted into pUC18 and confirmed to be a thermostable DNA polymerase gene by restriction mapping and DNA sequencing. The insert fragment was then combined into an expression vector, pBV221. This recombinant plasmid overexpressed a 94-kDa of recombinant protein in E. coli. A 100-mL E. coli. culture could yield 1.5 x 10(5) units of Taq DNA polymerase, which could be applied in the PCR.
Jin
C
C
Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
Liu
H
H
Yang
S
S
Zhang
S
S
D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D012194:RNA-Directed DNA Polymerase / Q000235:genetics; D019914:Taq Polymerase; D013696:Temperature
D004269:DNA, Bacterial; D019914:Taq Polymerase; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase