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 af...
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Published in | The Journal of biological chemistry Vol. 266; no. 14; pp. 9153 - 9160 |
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Main Authors | , , , |
Format | Journal Article |
Language | English |
Published |
Bethesda, MD
American Society for Biochemistry and Molecular Biology
15.05.1991
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Subjects | |
Online Access | Get full text |
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Summary: | 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. |
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Bibliography: | ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 |
ISSN: | 0021-9258 1083-351X |
DOI: | 10.1016/s0021-9258(18)31564-3 |