( fusion protein gave approximately 4 mg ...)New Yeast Cloning System for Producing Proteins with Native Amino Acid,,,Sequences

fusion protein gave approximately 4 mg per liter (data not shown). The GST-chicken calmodulin recombinant protein produced approximately 12 mg per liter of fusion protein. Figure 3 shows the one-column purification of the GST-calmodulin fusion protein and subsequent digestion with enterokinase.

Conclusions

The pESP LIC cloning and protein purification kits allow efficient and directional cloning of high-fidelity PCR products immediately downstream of the GST purification tag and the recognition site for enterokinase. Single-step purification of the recombinant GST fusion protein and removal of the purification tag by enterokinase cleavage yield polypeptides with completely native amino acid sequences. The ESP LIC cloning and protein purification kit includes all the necessary components for LIC cloning, expression in S.pombe and GST affinity purification.

Materials and Methods

For protein induction, the expression strain was inoculated into 10 ml of YES media and incubated at 30C overnight. This culture was used to inoculate another 10 ml of YES media and was incubated at 30C until the culture reached mid-log phase. The cells were collected by centrifugation, washed once with 50 ml of sterilized water, resuspended in 10 ml of EMM media with 25 M thiamine (repressed) or without thiamine (induced) and incubated at 30C for 18 to 20 hours. Crude lysates were prepared by washing cells in PBST buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄ and 1% Triton X-100), then lysing cells by vortexing with glass beads in PBST buffer containing protease inhibitors (1 mM PMSF, 1 g/ml aprotinin, 1 M pepstain A, 100 M leupeptin and 1 g/ml chymostatin). Soluble protein was bound to glutathi
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