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

s of the S. pombe host strain and reagents for protein induction and purification.

The pESP-1 Expression Vector

figure 1

Designed for expression in S. pombe, the pESP-1 vector (figure 1) is composed of the following parts: (1) a ColE1 origin of replication and an ampicillin resistance (Ampr) fragment, which allow vector replication and antibiotic selection in E. coli; (2) the ars1 fragment, which provides an origin of replication for the vector to replicate autonomously in S. pombe; (3) a LEU2-d gene from Saccharomyces cerevisiae, which serves as a selection marker for transforming the expression vector into S. pombe cells (strain SPQ01) and (4) the expression cassette, which contains the S. pombe nmt1 promoter, a translational start site, a GST protein-tag sequence, a multiple cloning site and the nmt1 transcription termination signal. Insertion of the gene of interest into the multiple cloning site results in an N-terminal fusion with the GST peptide, which facilitates one-step purification of the GST fusion protein.2

Figure 3

The nmt1 promoter of S. pombe is tightly repressed in the presence of thiamine (vitamin B1) in the growth medium and is highly activated upon its removal.3 When activated, the nmt1 promoter has been shown to be one of the strongest promoters in S. pombe.4 For this reason, the nmt1 promoter has been widely used to study gene function in S. pombe. Figure 3 shows Western blot analysis of the GST-calmodulin fusion protein in the pESP-1 vector that has been expressed in cells grown under
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