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A New C-Terminal GST Vector for Protein Production in S. pombe

l">, corresponds to nucleotide sequence 6968-6989. The reverse primer, 5-GATATTCCAAAAGAAGTCGAGTGGG-3, corresponds to nucleotide sequence 7235-7259. These primers can also be used to confirm the cloning junctions by sequencing.

Expression and purification of recombinant protein. For protein induction, the expression strain (SP-Q01 transformed with the pESP-3 vector containing the calmodulin gene) was inoculated into 5 ml of YES media and incubated at 30C overnight. A portion of the overnight culture was used to inoculate 10 ml of YES media (OD600=0.1). Four to five hours of further incubation at 30C were required for the culture to reach mid-logarithmic phase (OD600=0.5). The cells were collected by centrifugation at 1000 x g in a benchtop centrifuge and washed once with 50 ml of sterilized water. The cells were then resuspended into 10 ml of EMM media with 25 M thiamine (repressed) or without thiamine (induced) and grown at 30C for 18 to 20 hours. The cells were collected and washed once with PBST buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 and 1% Triton X-100), and the cell pellet was further resuspended in 500 l of PBST buffer containing protease inhibitors (1 mM PMSF, 1 g/ml aprotinin, 1 M pepstain A, 100 M leupeptin and 1 g/ml chymostatin). Five hundred micrograms of acid-washed glass beads (0.4- to 0.6-mm diameter) were added, and the cells were lysed by vortexing at 4C for 5 minutes. After centrifugation in a microcentrifuge for 5 minutes at 12,000 x g, the supernatant (crude lysate) was saved and stored at -70C for further purification and analyses. The GST-tagged fusion protein was purified as previously described.1

Acknowledgments

We would like to thank Wei-Ping Yang, Connie Hansen, Edward Arana
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