PURIFICATION AND CHARACTERIZATION OF THERMUS-CALDOPHILUS GK24 DNA-POLYMERASE
PURIFICATION AND CHARACTERIZATION OF THERMUS-CALDOPHILUS GK24 DNA-POLYMERASE
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DOI：
10.1111/j.1432-1033.1993.tb17905.x
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发表时间：
1993-05-15
期刊：
影响因子：
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通讯作者：
LEE, DS
中科院分区：
文献类型：
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作者：
PARK, JH;KIM, JS;LEE, DS
通过离子交换、凝胶过滤和亲和层析等色谱方法，将来自嗜热栖热菌 GK24 的热稳定性 DNA 聚合酶纯化至接近均质。通过 SDS/PAGE 和 Superose-12 凝胶过滤估算，纯化的酶在 75℃ 下的比活性为 8400 U/mg，分子量为 95 kDa。从pH、金属离子浓度和温度方面研究了反应条件。实验结果表明，Tca DNA 聚合酶在 75 ℃、pH 8.7 附近具有最大活性。该酶的N端序列与栖热菌(Taq)DNA聚合酶的N端序列高度相似，这与该酶具有5'-to-3'核酸外切酶活性而没有3'-to-5'核酸外切酶活性的事实相一致。使用 Tca DNA 聚合酶的基因扩增比使用 Taq DNA 聚合酶的基因扩增产生更长的产物。
A thermostable DNA polymerase from Thermus caldophilus GK24 was purified to near homogeneity by chromatographic methods, including ion-exchange, gel-filtration and affinity chromatography. The purified enzyme had a specific activity of 8400 U/mg at 75-degrees-C and a molecular mass of 95 kDa, estimated by SDS/PAGE and Superose-12 gel filtration. Reaction conditions were investigated in terms of pH, metal-ion concentration and temperature. Experimental results showed that T caldophilus (Tca) DNA polymerase had a maximum activity near pH 8.7 at 75-degrees-C. The N-terminal sequence of the enzyme was highly similar to that of Thermus aquaticus (Taq) DNA polymerase, which was consistent with the fact that the enzyme had 5'-to-3' exonuclease activity and no 3'-to-5' exonuclease activity. Gene amplification using Tca DNA polymerase resulted in longer products than amplification using Taq DNA polymerase.