Thermostable DNA-polymerase from the thermophilic archaeon microorganism Archaeoglobus fulgidus VC16 and its features.
A gene (No. AF0497 GenBank, USA) was cloned from the archaeon Archaeoglobus fulgidus strain found in the water of hot springs. This gene contains an open reading frame of 2346 base pairs which encodes a thermostable DNA-polymerase (762 amino acid residues). A recombinant protein Afu-pol with molecular weight of 94 kD was isolated in an Escherichia coli strain used as a producer and characterized. By site-directed mutagenesis in the afu-pol gene the amino acid residue Glu170 was replaced with Ala; this resulted in a complete loss of the 3;-5;-exonuclease activity of the enzyme. Thus, the Glu170 residue was suggested to be directly involved in formation of the 3;-5;-exonuclease site. Physicochemical features of the exodeficient enzyme form were studied, and the possible use of Afu(exo(-))-pol in the polymerase chain reaction is shown.
School of Chemistry, Lomonosov Moscow State University, Moscow, 119992 Russia. chalov@comp.chem.msu.ru
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Chalov
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Voronina
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Sergienko
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Lunin
D019943:Amino Acid Substitution; D019611:Archaeoglobus fulgidus / Q000201:enzymology* / Q000235:genetics; D001345:Autoradiography; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000378:metabolism; D019766:Genes, Archaeal; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000737:chemistry / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011189:Potassium Chloride / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature
D017931:DNA Primers; D011994:Recombinant Proteins; D011189:Potassium Chloride; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; D008274:Magnesium