HIGH-LEVEL PRODUCTION AND PURIFICATION OF BIOLOGICALLY-ACTIVE PROTEINS FROM BACTERIAL AND MAMMALIAN-CELLS USING THE TANDEM PGFLEX EXPRESSION SYSTEM

Because of the complexities involved in the regulation of gene express ion in Escherichia coli and mammalian cells, it is considered general practice to use different vectors for heterologous expression of recom binant proteins in these host systems. However, we have developed and report a shuttle vector system, pGFLEX, that provides high-level expre ssion of recombinant glutathione S-transferase (GST) fusion proteins i n E. coli and mammalian cells. pGFLEX contains the cytomegaloma virus (CMV) immediate-early promoter in tandem with the E. coli lacZpo syste m. The sequences involved in gene expression have been appropriately m odified to enable high-level production of fusion proteins in either c ell type. The pGFLEX expression system allows production of target pro teins fused to either the N or C terminus of the GST pi protein and pr ovides rapid purification of target proteins as either GST fusions or native proteins after cleavage with thrombin. The utility of this vect or in identifying and purifying a component of a multi-protein complex is demonstrated with cyclin A. The pGFLEX expression system provides a singular and widely applicable tool for laboratory or industrial pro duction of biologically active recombinant proteins in E. coli and mam malian cells. (C) 1997 Elsevier Science B.V.