( Versatile eukaryotic expressio...)A New C-Terminal GST Vector for Protein Production in S. pombe

Versatile eukaryotic expression and protein purification vector

Quinn Lu Tanya Hosfield
Stratagene

Stratagene has constructed and tested the new pESP-3 vector, which can be used for C-terminal fusion with glutathione-S-transferase (GST) and for protein production in Schizosaccharomyces pombe. After expression and purification of the GST fusion protein using the ESP yeast protein expression and purification system, the GST tag can be removed by proteolytic cleavage with thrombin or enterokinase.

Expression and purification of heterologous genes often involve fusion of the gene of interest with a purification tag at either the N-terminus or C-terminus. The Schistosoma japonicum GST gene product has been popularly used as a protein fusion tag for affinity purification of GST fusion proteins.¹ Stratagenes ESP yeast protein expression and purification system includes the pESP-1 and pESP-2 vectors for N-terminal tagging with GST.^2-4 The fission yeast S. pombe, which is used in the ESP system, possesses characteristics that closely resemble those of higher eukaryotic organisms regarding chromosome structure and function, cell cycle control and RNA splicing.⁵ Stratagenes ESP system features high-level protein production in S. pombe, thereby retaining the posttranslational modifications of eukaryotic proteins that may be critical for their structure and function.

The availability of an expression vector for C-terminal tagging with GST is desirable since the stability and activity of fusion proteins may vary with the location of the tag.^6-8 In addition, by tagging a protein at the C-terminus, only fully translated proteins will have the GST affinity tag, ensuring purification of full-length fusion proteins. Stratagene has constructed
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