Bacterial expression of the Saccharomyces cerevisiae ubiquitin-conjugating enzyme Ubc7.
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.
Yamazaki
R K
RK
Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Chau
V
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D000255:Adenosine Triphosphate / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008025:Ligases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D012995:Solubility; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism*; D014508:Urea / Q000494:pharmacology
D011994:Recombinant Proteins; D014452:Ubiquitins; D000255:Adenosine Triphosphate; D014508:Urea; C079437:UBE2L3 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D008025:Ligases