A plasmid system for high-level expression and in vitro processing of recombinant proteins.
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.
Pohlner
J
J
Max-Planck-Institut für Biologie, Infektionsbiologie, Tübingen, Germany.
Krämer
J
J
Meyer
T F
TF
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics; D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006639:Histidine; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D009532:Nickel; D009841:Oligonucleotides; D010447:Peptide Hydrolases / Q000378:metabolism; D010957:Plasmids / Q000235:genetics*; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000378:metabolism; D012697:Serine Endopeptidases
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D006639:Histidine; D009532:Nickel; D010447:Peptide Hydrolases; D012697:Serine Endopeptidases; C021433:IgA-specific serine endopeptidase