Purification of eukaryotic MutL homologs from Saccharomyces cerevisiae using self-cleaving affinity technology

Self-cleaving affinity technology is an effective tool for rapid purificati on of native sequence recombinant proteins overproduced in Escherichia coli . In this report, we describe the adaptation of this technology to purify D NA mismatch repair proteins overproduced in the eukaryote Saccharomyces cer evisiae. Mlh1 and Pms1 are homologs of the E. coli MutL protein that partic ipate in a variety of DNA transactions in cells, including correction of DN A replication errors, recombination, excision repair, and checkpoint contro l. Difficulties in preparing substantial quantities of highly purified MutL homologs have impeded descriptions of their biophysical and biochemical pr operties and mechanisms of action. To overcome this limitation, here we use self-cleaving affinity technology to purify to apparent homogeneity the ye ast Mlh1-Pms1 heterodimer and the individual yeast and human Mlh1 subunit. The availability of these proteins should accelerate an understanding of th eir multiple functions in mismatch repair and other DNA transactions. The g eneral approach is a valid alternative for simple, rapid purification of re combinant proteins in yeast when expression in bacteria is unsuitable. (C) 2001 Academic Press.