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

potentially toxic to the host cannot be overexpressed under constitutive expression conditions. Using the S. pombe expression system, we have purified 10 different proteins, including the rat c-Jun N-terminal kinase (JNK) and the human MEK1 gene products (figure 4). Yield for these proteins has varied from 1.0 mg per liter to 12.5 mg per liter. Some of these proteins, especially MEK1, had been difficult to produce in significant quantities in E. coli.

figure 4

Removing the GST Tag by Proteolytic Cleavage with Enterokinase or Thrombin

In many cases, GST tags do not interfere with the function of the proteins. Our functional assays indicated that the biological activities of the GST-calmodulin fusion protein and the GST-MEK1 fusion protein expressed in S. pombe were retained (data not shown). However, if desired, the GST tag can be removed by treating the purified fusion protein with either enterokinase or thrombin to cleave between the GST tag and the protein of interest (figure 1). Figure 5 shows an example of protease cleavage of a GST-MEK1 fusion protein. The GST tag was efficiently detached from the MEK1 moiety by digesting the purified GST-MEK1 fusion protein with enterokinase. The enterokinase site is often preferred for cleavage since the resulting product has fewer additional amino acids remaining at the N terminus. Alternatively, if the recombinant protein is cleaved with thrombin, the resulting polypeptide will contain the FLAG peptide at its N terminus, which could facilitate further analysis of the protein.


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