A T7 EXPRESSION VECTOR FOR PRODUCING N-TERMINAL AND C-TERMINAL FUSION PROTEINS WITH GLUTATHIONE-S-TRANSFERASE

The pGEX system for protein production in E. coli is widely used in molecular biology. A bacterial expression vector, pETGEXCT, which incorporates features of the pGEX and PET expression systems was designed. pETGEXCT allows the production of N- and C-terminal fusions to glutathione S-transferase (GST) under the tight control of the T7 promoter. Use of this vector can circumvent problems associated with unstable or inactive fusions to the N terminus of GST. Indeed, it is demonstrated that fusions to the N terminus of the eukaryotic DNA-binding protein, RSRFC4 cannot be tolerated. Fusion of RSRFC4 to the N terminus of GST in the pETGEXCT vector is a prerequisite to purify the RSRFC4 DNA-binding domain in an active form using glutathione-agarose affinity chromatography.