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Subcloning, Expression and Purification of Functional E. coli Nucleotide Excision Repair Protein UvrA Using IMPACT-CN System.

机译：使用IMPACT-CN系统进行功能性大肠杆菌核苷酸切除修复蛋白UvrA的亚克隆，表达和纯化。

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DNA in cells is constantly damaged by both endogenous and exogenous genotoxic agents. Nucleotide excision repair (NER) in Escherichia coli (E. coli) is one of the DNA repair systems that recognizes and removes a variety of DNA damage such as pyrimidine dimers, bulky chemical adducts, DNA intrastrand cross-links, etc. The genes responsible for E. coli NER incisions are UvrA, UvrB, and UvrC. Purification of UvrA, UvrB, and UvrC is essential for research to understand the molecular mechanisms of NER and carcinogenesis. Although UvrA has been successfully purified in our lab in the past, the experimental procedures were very time-consuming and technically challenging. In this study we employed IMPACT (Intein Mediated Purification with an Affinity Chitin-binding Tag) system to subclone the cDNA of UvrA and express and purify the recombinant UvrA protein by a single-column step. The purified UvrA protein was found to be fully functional in the UvrABC incision assay.

机译：内源性和外源性遗传毒性剂都会不断破坏细胞中的DNA。大肠杆菌（E. coli）中的核苷酸切除修复（NER）是一种DNA修复系统，可识别和消除各种DNA损伤，例如嘧啶二聚体，庞大的化学加合物，DNA内链交联等。大肠杆菌NER切口为UvrA，UvrB和UvrC。 UvrA，UvrB和UvrC的纯化对于研究了解NER和致癌作用的分子机制至关重要。尽管UvrA过去已在我们的实验室中成功纯化，但实验过程非常耗时且技术上具有挑战性。在这项研究中，我们采用IMPACT（具有亲和性几丁质结合标签的蛋白介导的纯化）系统亚克隆UvrA的cDNA，并通过单列步骤表达和纯化重组UvrA蛋白。发现纯化的UvrA蛋白在UvrABC切口测定中具有完全功能。

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作者
Lin, Cathy W.;
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作者单位

East Tennessee State University.;

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授予单位 East Tennessee State University.;
学科 Chemistry Molecular.
学位 M.S.
年度 2014
页码 48 p.
总页数 48
原文格式 PDF
正文语种 eng
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3. Over-expression in E. coli and purification of functional full-length murine small C-terminal domain phosphatase (SCP1, or Golli-interacting protein) q [J] . Sergio Jaramillo-Tatis, Vladimir V. Bamm, Kenrick A. Vassall, Protein Expression and Purification . 2014,第Null期

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Subcloning, Expression and Purification of Functional E. coli Nucleotide Excision Repair Protein UvrA Using IMPACT-CN System.

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