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

the GST gene. Cleavage of the fusion protein with enterokinase results in a protein with the FLAG epitope tag attached on its C-terminus; cleavage by thrombin removes both the GST sequence and the FLAG epitope.

Expression and Purification of Chicken Calmodulin

The chicken calmodulin gene was inserted into the Nde I and BamH I sites of the pESP-3 vector, serving as a test gene for protein expression and purification. The calmodulin gene primers were designed such that the calmodulin gene would be fused in frame at its C-terminus to the GST gene. Recombinant clones were identified by colony PCR analysis using vector-specific primers (the pESP-3 vector forward and reverse primers) that flank the MCS. A positive clone was chosen for further study.

Figure 2 Panel A

figures 2 Panel B

figures 2 Panel C

The pESP-3 vector containing the chicken calmodulin gene was transformed into the S. pombe strain SP-Q01 using a standard protocol.11 Figure 2 Panel A shows the expression of the calmodulin-GST fusion protein and purification by one-step chromatography using the glutathione-agarose beads1 contained in the ESP yeast protein expression and purification kit. The level of induced expression of the calmodulin-GST fusion protein is estimated to be 10% to 15% of the total soluble protein. Approximately 12.5 mg of the calmodulin-GST fusion protein was obtained from 1 liter of induced culture. After purification of the fusion protein, the GST tag was removed by incubating the fusion protein with either enterokinase or thrombin (figures 2 Panel B and Panel C). In similar experiments, the firefly luciferase
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