Purification and properties of Aquifex aeolicus DNA polymerase expressed in Escherichia coli.
The gene encoding Aquifex aeolicus (Aae) DNA polymerase was expressed under the control of the trp promoter on a high-copy plasmid, pTRPNS, in Escherichia coli. The expressed enzyme was purified 11-fold with a 13.8% yield and a specific activity of 2268.3 U mg(-1). The optimum pH of the enzyme was 6.8-7.2. The optimal concentrations of KCl and Mg(2+) were 20-30 mM and 4-5 mM, respectively. Aae DNA polymerase contained a double-strand-dependent 3'-->5' proofreading exonuclease activity but lacked any detectable 5'-->3' exonuclease activity.
Chang
J R
JR
Department of Genetic Engineering, Sungkyunkwan University, 300 Chunchon-dong, Jangan-Ku, Suwon 440-746, South Korea.
Choi
J J
JJ
Kim
H K
HK
Kwon
S T
ST
D002413:Cations, Divalent / Q000494:pharmacology; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics*; D005092:Exonucleases / Q000378:metabolism; D016946:Gram-Negative Aerobic Rods and Cocci / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature; D014169:Transformation, Bacterial
D002413:Cations, Divalent; D011994:Recombinant Proteins; D011189:Potassium Chloride; D004247:DNA; D004492:Edetic Acid; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases; D008274:Magnesium