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Purification and properties of Thermus filiformis DNA polymerase expressed in Escherichia coli
Author(s) -
Choi Jeong Jin,
Jung Seung Eun,
Kim HyunKyu,
Kwon SukTae
Publication year - 1999
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1111/j.1470-8744.1999.tb01154.x
Subject(s) - thermus aquaticus , thermus , escherichia coli , enzyme , microbiology and biotechnology , polymerase , dna , dna polymerase , plasmid , biology , polymerase chain reaction , enzyme assay , biochemistry , molecular mass , chemistry , gene , thermophile
The gene encoding Thermus filiformis ( Tfi ) DNA polymerase was expressed under the control of the tac promoter on a high‐copy plasmid, pJR, in Escherichia coli . The Tfi DNA polymerase was purified by using heat treatment and DEAE‐Sephacel column chromatography. The purified enzyme had a molecular mass of 92 kDa, as estimated by SDS/PAGE. The optimum pH and temperature of the enzyme were 8.4–9.0 and 70–72.5 °C respectively. The half‐life of the enzyme at 94 °C was approx. 40 min. The enzyme was activated by the bivalent cations, Mg 2+ and Mn 2+ , and was inhibited by EDTA. The optimal Mg 2+ concentration of the enzyme was 4 mM. The optimal conditions for the PCR reaction were slightly different from those for the enzyme activity except for the optimal Mg 2+ concentration. Low concentrations of KCl had no effect on either the enzymic activity or the PCR amplification. The result of the PCR experiment with the enzyme indicates that Tfi DNA polymerase might be useful in DNA amplification.