[Cloning of the gene for thermostable Thermus aquaticus YT1 DNA polymerase and its expression in Escherichia coli].
Using the phasmid vector pSL5, the genomic DNA fragment of T. aquaticus YT1 which contained the thermostable DNA polymerase (Taq-polymerase) gene was cloned. The BglII fragment of this genome locus was subcloned in the BamHI site of the pUC19 plasmid. To optimize the Taq-polymerase gene expression in E. coli cells, the gene was cloned in the correct reading frame regarding the initiation ATG codon of the pPR-TGATG-1 expression vector. The gene expression in this vector was controlled by the phage lambda PR promoter and the temperature-sensitive phage lambda repressor. We used PCR to amplify the short 5'-end fragment of the Taq-polymerase gene coding for the part into which an artificial SacI site was introduced. This site has been used for cloning the PCR product into the pPR-TGATG-1 vector, and the missing gene part was cloned into the KpnI site of the PCR product from the natural cloned gene. The cells of the E. coli PVG-A1 strain, which was obtained in the end, expressed efficiently the Taq-polymerase gene at the nonpermissive temperature. The content of the recombinant Taq-polymerase in the cells was about 1-2% of total proteins. The purified nearly homogeneous Taq-polymerase amplified efficiently in the PCR DNA fragments up to 5.5 kb long and was useful in DNA sequencing the by Sanger method. The half-life of the purified Taq polymerase was about 60 min at 95 degrees C, it was active for at least 65 standard PCR circles. The specific activity of recombinant enzyme preparations was about 180-200,000 units per mg of protein. The E. coli PVG-A1 strain enables one to isolate up to 500,000 units of purified enzyme from 2 l of bacterial culture.
Patrushev
L I
LI
Valiaev
A G
AG
Golovchenko
P A
PA
Vinogradov
S V
SV
Chikindas
M L
ML
Kiselev
V I
VI
D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D019914:Taq Polymerase; D013824:Thermus / Q000201:enzymology*
D017931:DNA Primers; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase
Klonirovanie gena termostabil'noĭ DNK-polimerazy Thermus aquaticus YT1 i ego ékspressiia v kletkakh Escherichia coli.