嗜热栖热菌DNA聚合酶的纯化、结晶及初步X射线晶体学分析
Purification, crystallization and preliminary X-ray crystallographic analysis of Pyrococcus furiosus DNA polymerase.
作者信息
Goldman S, Kim R, Hung L W, Jancarik J, Kim S H
机构信息
Physical Biosciences Division of Lawrence Berkeley National Laboratory and Department of Chemistry, University of California, Berkeley, CA 94720, USA.
出版信息
Acta Crystallogr D Biol Crystallogr. 1998 Sep 1;54(Pt 5):986-8. doi: 10.1107/s0907444998000353.
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.
嗜热古菌激烈火球菌（Pyrococcus furiosus）的DNA聚合酶基因已被克隆，其蛋白质在大肠杆菌中过量表达以产生一种活性酶。纯化后的蛋白质通过气相扩散法，从含有0.08 M硫酸铵、0.05 M二甲胂酸钠（pH 6.5）、0.15%（v/v）NP40、0.05%（v/v）吐温20和4.5%（w/v）聚乙二醇6000的溶液中结晶。正交晶体的晶胞参数为a = 92.5、b = 125.4、c = 192.1 Å；α = β = γ = 90°。这些晶体在1.08 Å的同步辐射源上衍射能力超过4 Å。
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