A plasmid system for high-level expression and in vitro processing of recombinant proteins

Abstract

A novel plasmid expression system has been constructed that combines two useful functions: it facilitates single-step affinity purification of cytoplasmically overproduced fusion proteins and the in vitro processing of fusions with IgA protease (Igase). The significant features directing the high expression rate of pEV41-based gene fusions in Escherichia coli are the lambda pL promoter for temperature-regulated transcription and the translation initiation region of the bacteriophage MS2 polymerase gene including a downstream box (db) within the first few codons of the open reading frame. Fusion proteins generated with this system contain a short N-terminal carrier peptide allowing convenient affinity purification by means of the His6 peptide. As exemplified by the production of the variable heavy (VH) and light (VL)-chain domains of a monoclonal antibody, the fusion proteins can be specifically processed with Igase either in purified form or simply by incubation with the culture medium of recombinant E. coli [pJP10] cells. Chemical cross-linking of processed VH and VL domains resulted in a recombinant antibody Fv fragment that can specifically bind to its antigen.