McDonald W F, Traktman P
Department of Microbiology, Cornell University Medical College, New York, New York 10021.
Protein Expr Purif. 1994 Aug;5(4):409-21. doi: 10.1006/prep.1994.1059.
We have overexpressed the vaccinia virus DNA polymerase using the hybrid vaccinia virus/T7 expression system. Accumulation of the DNA polymerase to levels as high as 10% of the total protein was observed following coinfection of BSC40 cells with the appropriate vaccinia recombinants. Although the DNA polymerase produced at 37 degrees C was largely insoluble, 25% of the recombinant protein could be recovered as soluble protein when infected cultures were maintained at 32 degrees C. Starting with cytoplasmic lysates of coinfected cells, a rapid and reproducible purification protocol that yielded apparently homogeneous preparations of the DNA polymerase after four chromatographic steps was established. Typically, 0.3 mg of purified DNA polymerase was obtained from 27 mg of total protein within 10 h after harvesting infected cells. As was previously described for the DNA polymerase purified from vaccinia-infected cells (Challberg and Englund, J. Biol. Chem., 254, 7812-7819, 1979), the purified recombinant enzyme displayed both polymerase and 3'-5' exonuclease activities but lacked detectable 5'-3' exonuclease activity. Kinetic analysis of nucleotide incorporation catalyzed by the vaccinia enzyme revealed apparent Km values of 0.9, 2.9, 4.0, and 2.7 microM for dGTP, dATP, TTP, and dCTP, respectively.
我们使用痘苗病毒/T7杂交表达系统对痘苗病毒DNA聚合酶进行了过表达。在用合适的痘苗重组体共感染BSC40细胞后，观察到DNA聚合酶积累至总蛋白的10%之高。尽管在37℃产生的DNA聚合酶大部分不溶，但当感染培养物维持在32℃时，25%的重组蛋白可作为可溶性蛋白回收。从共感染细胞的细胞质裂解物开始，建立了一种快速且可重复的纯化方案，经过四个色谱步骤后可得到明显均一的DNA聚合酶制剂。通常，在收获感染细胞后10小时内，从27mg总蛋白中可获得0.3mg纯化的DNA聚合酶。如先前对从痘苗感染细胞中纯化的DNA聚合酶的描述（Challberg和Englund，《生物化学杂志》，254，7812 - 7819，1979），纯化的重组酶兼具聚合酶和3'-5'核酸外切酶活性，但缺乏可检测到的5'-3'核酸外切酶活性。对痘苗酶催化的核苷酸掺入的动力学分析显示，dGTP、dATP、TTP和dCTP的表观Km值分别为0.9、2.9、4.0和2.7μM。
