Using Schizosaccharomyces pombe as a host for expression and purification of eukaryotic proteins.
We have established a eukaryotic protein expression and purification system by using the yeast Schizosaccharomyces pombe as the host and the glutathione S-transferase (GST) as a protein purification tag. This system provides opportunities for rapid, inexpensive, and high yield production of proteins in a eukaryotic organism. Unlike E. coli, S. pombe provides for post-translational modifications of the proteins, which are often critical for the structure and function of eukaryotic proteins. Two vectors have been constructed for protein expression in S. pombe, pESP-1 and pESP-2. Both vectors use the nmt1 promoter for constitutive or induced expression of the gene of interest. Expressed GST-tagged proteins are easily and rapidly purified using glutathione agarose beads. The GST tag can be removed from the fusion proteins by treatment with either the thrombin or enterokinase protease. Proteins expressed from the pESP-2 vector will yield native amino acid sequence when the GST tag is removed by treatment with enterokinase. Nine proteins have been purified by using the system with yields ranging from 1.0 mg/l to 12.5 mg/l of induced culture.
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Stratagene Cloning Systems, La Jolla, CA 92037, USA. qlu@stratagene.com
Bauer
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JC
Greener
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000096:biosynthesis / Q000302:isolation & purification; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics; D017238:Genes, Helminth; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D008969:Molecular Sequence Data; D009842:Oligopeptides; D010455:Peptides; D011401:Promoter Regions, Genetic; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000302:isolation & purification; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D012549:Schistosoma japonicum / Q000201:enzymology / Q000235:genetics; D012568:Schizosaccharomyces; D029702:Schizosaccharomyces pombe Proteins; D016324:Sequence Tagged Sites
D005656:Fungal Proteins; C093953:NMT1 protein, S pombe; D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D029702:Schizosaccharomyces pombe Proteins; C085706:FLAG peptide; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases