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Reference: Yamazaki RK and Chau V (1996) Bacterial expression of the Saccharomyces cerevisiae ubiquitin-conjugating enzyme Ubc7. Protein Expr Purif 7(1):122-7

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Abstract

The coding sequence for the yeast ubiquitin-conjugating enzyme Ubc7 was obtained by PCR from Saccharomyces cerevisiae genomic DNA. This sequence was placed in a plasmid containing the lambdaPL promoter and was used for temperature-regulated expression in Escherichia coli. The expressed 18-kDa protein was isolated in the inclusion body fraction from bacterial lysates, in contrast to the soluble nature of other yeast ubiquitin-conjugating enzymes expressed in E. coli. Selective solubilization of the protein using 5 M urea followed by dialysis, MonoQ FPLC, and Superdex-75 FPLC yielded electrophoretically pure Ubc7 protein. The purified protein was enzymatically active as determined by formation of enzyme-linked thiolester with ubiquitin. The ability of Ubc7 protein to regain enzymatic activity after urea denaturation appears to be attributable to the stable core alpha/beta folded structure common to the ubiquitin-conjugating enzymes whose structures have been determined to date.

Reference Type
Journal Article | Research Support, U.S. Gov't, P.H.S.
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Yamazaki RK, Chau V
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