title: High-level production and purification of biologically active proteins from bacterial and mammalian cells using the tandem pGFLEX expression system.
abstract: Because of the complexities involved in the regulation of gene expression in Escherichia coli and mammalian cells, it is considered general practice to use different vectors for heterologous expression of recombinant proteins in these host systems. However, we have developed and report a shuttle vector system, pGFLEX, that provides high-level expression of recombinant glutathione S-transferase (GST) fusion proteins in E. coli and mammalian cells. pGFLEX contains the cytomegaloma virus (CMV) immediate-early promoter in tandem with the E. coli lacZpo system. The sequences involved in gene expression have been appropriately modified to enable high-level production of fusion proteins in either cell type. The pGFLEX expression system allows production of target proteins fused to either the N or C terminus of the GST pi protein and provides rapid purification of target proteins as either GST fusions or native proteins after cleavage with thrombin. The utility of this vector 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 production of biologically active recombinant proteins in E. coli and mammalian cells.
authors.affiliation: McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706, USA.
authors.forename: H T
authors.initials: HT
authors.lastname: Manoharan
authors.forename: J
authors.initials: J
authors.lastname: Gallo
authors.forename: A M
authors.initials: AM
authors.lastname: Gulick
authors.forename: W E
authors.initials: WE
authors.lastname: Fahl
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D003001:Cloning, Molecular / Q000379:methods; D003587:Cytomegalovirus / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D017781:Genes, Immediate-Early; D005982:Glutathione Transferase / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics*
chemical_list: D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase