Purification, crystallization and preliminary X-ray crystallographic analysis of Pyrococcus furiosus DNA polymerase.
DNA polymerase gene from the hyperthermophilic Archaeon Pyrococcus furiosus has been cloned and the protein overexpressed in Escherichia coli to produce an active enzyme. The purified protein was crystallized from 0.08 M ammonium sulfate, 0.05 M Na-cacodylate, pH 6.5, 0.15%(v/v) NP40, 0.05%(v/v) Tween 20 and 4.5%(w/v) polyethylene glycol 6000 by the vapour-diffusion method. The orthorhombic crystals had unit-cell dimensions of a = 92.5, b = 125.4, c = 192.1 A; alpha = beta = gamma = 90 degrees. The crystals diffracted beyond 4 A on a 1.08 A synchrotron radiation source.
Goldman
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Physical Biosciences Division of Lawrence Berkeley National Laboratory and Department of Chemistry, University of California, Berkeley, CA 94720, USA.
Kim
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R
Hung
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LW
Jancarik
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Kim
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SH
D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000302:isolation & purification; D011487:Protein Conformation; D020027:Pyrococcus furiosus / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification
D011993:Recombinant Fusion Proteins; C072146:Pfu DNA polymerase; D004259:DNA-Directed DNA Polymerase