Hall M C, Kunkel T A
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.
Protein Expr Purif. 2001 Mar;21(2):333-42. doi: 10.1006/prep.2000.1379.
Self-cleaving affinity technology is an effective tool for rapid purification of native sequence recombinant proteins overproduced in Escherichia coli. In this report, we describe the adaptation of this technology to purify DNA mismatch repair proteins overproduced in the eukaryote Saccharomyces cerevisiae. Mlh1 and Pms1 are homologs of the E. coli MutL protein that participate in a variety of DNA transactions in cells, including correction of DNA replication errors, recombination, excision repair, and checkpoint control. Difficulties in preparing substantial quantities of highly purified MutL homologs have impeded descriptions of their biophysical and biochemical properties and mechanisms of action. To overcome this limitation, here we use self-cleaving affinity technology to purify to apparent homogeneity the yeast Mlh1--Pms1 heterodimer and the individual yeast and human Mlh1 subunit. The availability of these proteins should accelerate an understanding of their multiple functions in mismatch repair and other DNA transactions. The general approach is a valid alternative for simple, rapid purification of recombinant proteins in yeast when expression in bacteria is unsuitable.
自切割亲和技术是一种用于快速纯化在大肠杆菌中过量表达的天然序列重组蛋白的有效工具。在本报告中，我们描述了该技术的适应性应用，用于纯化在真核生物酿酒酵母中过量表达的DNA错配修复蛋白。Mlh1和Pms1是大肠杆菌MutL蛋白的同源物，它们参与细胞中的多种DNA事务，包括DNA复制错误的校正、重组、切除修复和检查点控制。制备大量高度纯化的MutL同源物的困难阻碍了对其生物物理和生化特性以及作用机制的描述。为克服这一限制，我们在此使用自切割亲和技术将酵母Mlh1-Pms1异二聚体以及单个酵母和人Mlh1亚基纯化至表观均一性。这些蛋白的可得性应能加速对它们在错配修复和其他DNA事务中的多种功能的理解。当在细菌中表达不合适时，该通用方法是在酵母中简单、快速纯化重组蛋白的有效替代方法。
