Purification of eukaryotic MutL homologs from Saccharomyces cerevisiae using self-affinity technology.
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.
Hall
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Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.
Kunkel
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D048868:Adaptor Proteins, Signal Transducing; D000251:Adenosine Triphosphatases; D001426:Bacterial Proteins / Q000737:chemistry*; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002686:Chitin / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D002847:Chromatography, Agarose; D019281:Dimerization; D029968:Escherichia coli Proteins; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008970:Molecular Weight; D000070957:MutL Protein Homolog 1; D000070956:MutL Proteins; D009363:Neoplasm Proteins / Q000302:isolation & purification; D009687:Nuclear Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013997:Time Factors
D048868:Adaptor Proteins, Signal Transducing; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D005656:Fungal Proteins; C088859:MLH1 protein, S cerevisiae; C501486:MLH1 protein, human; C074770:MutL protein, E coli; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; C088858:PMS1 protein, S cerevisiae; C088996:PMS1 protein, human; D021122:Protein Subunits; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D002686:Chitin; D000251:Adenosine Triphosphatases; D000070957:MutL Protein Homolog 1; D000070956:MutL Proteins