title: Protein expression using cotranslational fusion and cleavage of ubiquitin. Mutagenesis of the glutathione-binding site of human Pi class glutathione S-transferase.
abstract: Expression of cloned genes in prokaryotes such as Escherichia coli is a widely used technique in both basic research and biotechnology. Despite the availability of several E. coli expression vector systems, adequate levels of expression may not be achieved. Expressing proteins as fusions to the highly conserved eukaryotic protein ubiquitin has been reported by several investigators to enhance protein yield in both bacterial and eukaryotic systems. We have modified this technique by the co-expression in E. coli of a ubiquitin-fusion protein and the Saccharomyces cerevisiae ubiquitin-specific protease Ubp2. This allows the co-translational cleavage of engineered ubiquitin-fusion proteins expressed in E. coli. This system was used to express a human Pi class glutathione S-transferase (GST) GSTP1 as well as two mutant GSTP1 derivatives, Trp39-->Cys and Gln52-->Glu. The yield of these enzymes was improved 40-fold by using the ubiquitin-fusion/co-translational cleavage technique, and no uncleaved product was detected. The Trp39-->Cys mutant was totally devoid of GST activity, while the activity of the Gln52-->Glu mutant was reduced to 6% of wild-type GSTP1-1. As both of the mutated residues map within the glutathione-binding site, the reduced GST activity is consistent with a marked reduction in glutathione binding ability.
authors.affiliation: Molecular Genetics Group, John Curtin School of Medical Research, Australian National University, Canberra.
authors.forename: R T
authors.initials: RT
authors.lastname: Baker
authors.forename: S A
authors.initials: SA
authors.lastname: Smith
authors.forename: R
authors.initials: R
authors.lastname: Marano
authors.forename: J
authors.initials: J
authors.lastname: McKee
authors.forename: P G
authors.initials: PG
authors.lastname: Board
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004252:DNA Mutational Analysis; D010450:Endopeptidases / Q000096:biosynthesis / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D005978:Glutathione / Q000378:metabolism*; D005982:Glutathione Transferase / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis* / Q000145:classification / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010920:Placenta / Q000201:enzymology; D010957:Plasmids / Q000235:genetics; D011247:Pregnancy; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013329:Structure-Activity Relationship; D014452:Ubiquitins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; D005982:Glutathione Transferase; D010450:Endopeptidases; C059237:ubiquitin-Nalpha-protein hydrolase; D005978:Glutathione