Purification and characterization of the Saccharomyces cerevisiae DNA polymerase delta overproduced in Escherichia coli.
Purification and characterization of the Saccharomyces cerevisiae DNA polymerase delta overproduced in Escherichia coli.
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DOI：
10.1016/s0021-9258(18)54030-8
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发表时间：
1993-01
期刊：
影响因子：
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通讯作者：
W. C. Brown;J. Duncan;J. Campbell
中科院分区：
文献类型：
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作者：
W. C. Brown;J. Duncan;J. Campbell
为了进一步确定酵母DNA聚合酶δ的酶性质，将在大多数克隆载体中表达对细菌具有高毒性的酿酒酵母POL3基因克隆到新的T7表达载体（W. C.布朗和J. L.坎贝尔，提交出版），其允许在细菌中有效过表达。从3g细胞获得15mg聚合酶。由于蛋白质以不溶性形式产生，为了获得活性聚合酶，用尿素溶解包涵体。在尿素存在下纯化DNA聚合酶δ（124 kDa），然后通过对含有降低浓度的尿素的缓冲液透析使其复性。复性的最佳蛋白浓度为5 μ g/ml。通过几个标准获得的酶是可比的，从酵母：比活性，电泳迁移率，模板偏好，抑制剂的敏感性，和持续合成能力。电泳迁移率表明，与DNA聚合酶α不同，聚合酶δ在酵母中不经过后修饰。针对来自细菌的全长DNA聚合酶delta提出了多克隆抗体，并且在蛋白质印迹上显示出与从酵母中纯化的蛋白质交叉反应。复性的蛋白质还表现出外切核酸水解活性。对该核酸酶的进一步检查确定其为具有校正活性特征的3 '至5'外切核酸酶。这种核酸酶在高度纯化的细菌聚合酶中的存在提供了早期遗传证据的生物化学证实（Simon，M.，乔特湖和Faye，G.（1991）EMBO J. 10，2165 - 2170），其表明DNA聚合酶δ的核心催化亚基含有内在的3 '至5'外切核酸酶。
In order to further define the enzymatic properties of yeast DNA polymerase delta, the Saccharomyces cerevisiae POL3 gene, whose expression is highly toxic to bacteria in most cloning vectors, was cloned into a new T7 expression vector (W. C. Brown and J. L. Campbell, submitted for publication) which allowed efficient overexpression in bacteria. Fifteen mg of polymerase were obtained from 3 g of cells. Since the protein is produced in insoluble form, to obtain active polymerase, inclusion bodies were solubilized with urea. DNA polymerase delta (124 kDa) was purified in the presence of urea and then renatured by dialysis against buffers containing decreasing concentrations of urea. Optimal protein concentration for refolding was 5 micrograms/ml. By several criteria the enzyme obtained is comparable with that from yeast: specific activity, electrophoretic mobility, template preference, sensitivity to inhibitors, and processivity. The electrophoretic mobility suggests that, unlike DNA polymerase alpha, polymerase delta is not posttranslationally modified in yeast. Polyclonal antibody was raised against the full-length DNA polymerase delta from bacteria and shown to cross-react with the protein purified from yeast on protein blots. The renatured protein also exhibits an exonucleolytic activity. Further examination of this nuclease determined it to be a 3' to 5' exonuclease with the characteristics of a proofreading activity. The presence of this nuclease in the highly purified bacterial polymerase provides biochemical confirmation of earlier genetic evidence (Simon, M., Giot, L., and Faye, G. (1991) EMBO J. 10, 2165-2170) that suggested that DNA polymerase delta's core catalytic subunit contains an intrinsic 3' to 5' exonuclease.