Bacterial expression of the Saccharomyces cerevisiae ubiquitin- conjugating enzyme Ubc7

Russell K. Yamazaki, Vincent Chau

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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 λP(L) 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 he attributable to the stable core α/β folded structure common to the ubiquitin-conjugating enzymes whose structures have been determined to date.

Original languageEnglish (US)
Pages (from-to)122-127
Number of pages6
JournalProtein Expression and Purification
Volume7
Issue number1
DOIs
Publication statusPublished - Feb 1996

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All Science Journal Classification (ASJC) codes

  • Biotechnology

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