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- W2354024723 abstract "Objective To express and purify Tgo DNA polymerase and study its amplificatioin performance.Methods The gene encoding Tgo DNA polymerase was amplified from Thermococcus gorgonarius by PCR and cloned into vector pET101 containing His-Tag target sequence.The constructed recombinant plasmid pET101-Tgo was transformed to E.coli BL21 Star(DE3)for expression under induction of IPTG.The expressed protein was purified,analyzed by SDS-PAGE,and determined for enzyme activity using standard pfu as control.Tgo and pfu were amplified by PCR using plasmid pUC19 as template,and their proliferation efficacies were compared.The fidelity of amplification of Tgo was verified by modified blue-white selection.Results The Tgo DNA polymerase gene at a length of about 2 300 bp was amplified by PCR,of which the amino acid sequence deduced based on the nucleotide sequence was consistent with that reported in GenBank.The expressed protein was in a soluble form with a relative molecular mass of about 89 000,of which the expression level was 350 μg/g E.coli,and the enzyme activity after storage in 50% glycerin was about 5 U/μl.Both the yield and fidelity of amplification of Tgo were equivalent to those of pfu.Conclusions Tgo DNA polymerase was successfully expressed and purified,of which the amplification performance was equivalent to that of pfu." @default.
- W2354024723 created "2016-06-24" @default.
- W2354024723 creator A5032682280 @default.
- W2354024723 date "2010-01-01" @default.
- W2354024723 modified "2023-09-25" @default.
- W2354024723 title "Expression,Purification and Amplification Performance of Tgo DNA Polymerase" @default.
- W2354024723 hasPublicationYear "2010" @default.
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