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

the pESP-3 vector for C-terminal tagging of a protein of interest with GST. The pESP-3 vector can also be used to express proteins without a tag for in vivo analysis of gene function.

The pESP-3 Vector

figure 1

The pESP-3 vector (figure 1) is a derivative of the pESP-2 vector. Similar to the pESP-1 and pESP-2 vectors, the new pESP-3 vector contains the following features: (1) a ColE1 origin of replication and an ampicillin-resistance gene (ampr), allowing vector replication and antibiotic selection in E. coli; (2) the ars1 fragment, providing the origin of replication for the vector to replicate autonomously in S. pombe; (3) a LEU2-d gene from Saccharomyces cerevisiae, serving as a selection marker for transforming the expression vector into S. pombe cells (strain SP-Q01, leu1-32h-) and (4) the expression cassette, containing the S. pombe nmt1 promoter, a translation start site, a multiple cloning site (MCS), a GST protein-tag sequence and the nmt1 transcription termination signal. 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.9 When activated, the nmt1 promoter has been shown to be one of the strongest promoters in S. pombe.10

When a gene of interest is inserted into the MCS of the pESP-3 vector, a C-terminal fusion with the GST peptide (~27 kDa) results, which facilitates one-step purification of the GST fusion protein.1 In order to provide convenient removal of the GST tag after purification, a sequence coding for the recognition and cleavage sites for enterokinase and thrombin have been engineered between the MCS and
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