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Yeast Protein Production System Features High Yields and One-Step,,,Purification

repressed and induced conditions. The fusion protein cannot be detected in cells when the promoter is repressed and is abundant in cells when the promoter is induced. In addition to inducing expression of the gene product of interest, constitutive high-level expression is possible by growing the clones in media without thiamine.

The pESP-1 vector also contains DNA sequences coding for amino acid residues that are specifically recognized and cleaved by enterokinase and thrombin. These sites are located between the GST tag and the multiple cloning site (figure 1). Insertion into the multiple cloning site results in fusion proteins that can be detached from the GST tag with either protease. Cleavage by thrombin results in a fusion protein having the FLAG peptide (DYKDDDDK)5 at its N terminus; antibodies against the FLAG peptide are available from Stratagene.

Establishing the Expression Strain

The gene of interest is inserted into the multiple cloning site of the pESP-1 vector and is fused in frame to the upstream GST gene. Successful cloning can be confirmed by sequence analysis. To establish the expression strain, the verified clone is then transformed into the competent cells of S. pombe host strain SP-Q01 ((leu1-32h ) that are included in the kit. These competent cells provide optimal transformation conditions. Transformants can be identified by their ability to grow on minimal medium (EMM) agar plates supplemented with 25 M thiamine. The expression strain can be stored at 70C and used for multiple experiments.

Induction and Purification of GST-Tagged Fusion Proteins

To induce expression of the protein of interest, the S. pombe expression strain is first grown to mid-logarithmic phase in the vegetative growth medium (YES medium), which prov
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Sterile concentration of Dithiothreitol (DTT) solution in phosphate buffer (pH 7.0). Dilute contents in one vial (10- fold) to 100 ml. For use in liquefying sputum samples....
5 minutes from gel band to purified DNA...
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