A modular set of prokaryotic and eukaryotic expression vectors.
A modular series of versatile expression vectors is described for improved affinity purification of recombinant fusion proteins. Special features of these vectors include (i) serial affinity tags (hexahistidine-GST) to yield extremely pure protein even with very low expression rates, (ii) highly efficient proteolytic cleavage of affinity tags under a variety of conditions by hexahistidine-tagged tobacco etch virus (TEV) protease, (iii) PCR cloning design that results in a product of proteolytic cleavage with only one (a single glycine) or two (gly-ala) amino acids at the N-terminus of the protein, and (iv) expression in either Escherichia coli or Saccharomyces cerevisiae. In addition, singly hexahistidine-tagged proteins can be produced for purification under denaturing conditions and some vectors allow addition of five amino acid kinase recognition sites for easy radiolabeling of proteins. To illustrate the use of these vectors, all regulatory components of the yeast GAL regulon, rather than abundant highly soluble proteins, were produced and purified under native or denaturing conditions, and their biological activity was confirmed.
Melcher
K
K
Institute for Microbiology, Biocenter, University of Frankfurt, Marie-Curie-Strasse 9, N250, Frankfurt/M., 60439, Germany. K.Melcher@em.uni-frankfurt.de
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D004268:DNA-Binding Proteins; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
D017931:DNA Primers; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D005982:Glutathione Transferase