Vectors for Cu(2+)-inducible production of glutathione S-transferase-fusion proteins for single-step purification from yeast.
We describe six new yeast episomal vectors which encode glutathione S-transferase (GST) affinity tags. These allow for the production of GST-fusion proteins in Saccharomyces cerevisiae under the control of the CUP1 promoter. Affinity chromatography with glutathione-Sepharose permits convenient purification of the fusion protein from a yeast lysate. The presence of a protease cleavage site facilitates subsequent removal of the GST tag. The expression and single-step purification of both GST and a functional GST-metallothionein fusion from yeast are shown as an example of the application of these vectors.
Ward
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Biomolecular Research Institute, Parkville, Victoria, Australia.
Castelli
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Macreadie
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Azad
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D003300:Copper / Q000494:pharmacology*; D004790:Enzyme Induction / Q000187:drug effects; D004926:Escherichia coli / Q000378:metabolism; D015951:Factor Xa / Q000235:genetics; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D008668:Metallothionein / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D015800:Protozoan Proteins / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D012549:Schistosoma japonicum / Q000201:enzymology* / Q000235:genetics; D013917:Thrombin / Q000235:genetics
D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D003300:Copper; D008668:Metallothionein; D005982:Glutathione Transferase; D013917:Thrombin; D015951:Factor Xa