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

The pGEX system for protein production in E. coli is widely used in mo lecular biology. A bacterial expression vector, pETGEXCT, which incorp orates features of the pGEX and PET expression systems was designed. p ETGEXCT allows the production of N- and C-terminal fusions to glutathi one S-transferase (GST) under the tight control of the T7 promoter. Us e of this vector can circumvent problems associated with unstable or i nactive fusions to the N terminus of GST. Indeed, it is demonstrated t hat fusions to the N terminus of the eukaryotic DNA-binding protein, R SRFC4 cannot be tolerated. Fusion of RSRFC4 to the N terminus of GST i n the pETGEXCT vector is a prerequisite to purify the RSRFC4 DNA-bindi ng domain in an active form using glutathione-agarose affinity chromat ography.