A rapid, efficient method for purifying DNA-binding proteins. Denaturation-renaturation chromatography of human and yeast mitochondrial extracts.
We describe a novel method for the purification of DNA-binding proteins. Isolated mitochondria were lysed in boiling sodium dodecyl sulfate-containing buffer, the extracts were chromatographed on hydroxylapatite in the presence of sodium dodecyl sulfate, and DNA-binding activities were identified after adding a large excess of nonionic detergent (Triton X-100) and assaying fractions by a gel retardation procedure. Fractions containing DNA-binding activity were bulk renatured and chromatographed on phosphocellulose in the presence of Triton X-100. When applied to human mitochondria, the technique resulted in the purification to homogeneity of fully functional mitochondrial transcription factor 1 (mtTF1), the major activator of mammalian mitochondrial transcription. Moreover, the yield of mtTF1 purified by this method was at least 25 times higher than that obtained by conventional nondenaturing chromatographies. When yeast mitochondria were subjected to the same protein isolation scheme, a 19-kilodalton putative yeast homologue of mtTF1 was purified to homogeneity. These results suggest that the denaturation-renaturation approach may be a valuable general method for the identification and high yield purification of DNA-binding proteins.
Fisher
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RP
Department of Developmental Biology, Stanford University School of Medicine, California 94305-5427.
Lisowsky
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T
Breen
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GA
Clayton
D A
DA
D000595:Amino Acid Sequence; D002460:Cell Line; D002845:Chromatography / Q000379:methods; D004272:DNA, Mitochondrial / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification*; D017886:Durapatite; D005656:Fungal Proteins / Q000302:isolation & purification; D006801:Humans; D006882:Hydroxyapatites / Q000737:chemistry; D008928:Mitochondria / Q000737:chemistry; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D011489:Protein Denaturation; D012441:Saccharomyces cerevisiae / Q000032:analysis; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D014157:Transcription Factors / Q000737:chemistry / Q000302:isolation & purification*
D004272:DNA, Mitochondrial; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D006882:Hydroxyapatites; D024101:Mitochondrial Proteins; D009687:Nuclear Proteins; C499631:TFAM protein, human; D014157:Transcription Factors; C422562:mitochondrial transcription factor A; D012967:Sodium Dodecyl Sulfate; D017886:Durapatite