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.
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Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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Site | Modification | Modifier | Source | Reference |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Assay | Annotation | Action | Modification | Source | Reference | Note |
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Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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