title: N(pro) fusion technology to produce proteins with authentic N termini in E. coli.
abstract: We describe a prokaryotic expression system using the autoproteolytic function of N(pro) from classical swine fever virus. Proteins or peptides expressed as N(pro) fusions are deposited as inclusion bodies. On in vitro refolding by switching from chaotropic to kosmotropic conditions, the fusion partner is released from the C-terminal end of the autoprotease by self-cleavage, leaving the target protein with an authentic N terminus. A tailor-made N(pro) mutant called EDDIE, with increased in vitro and decreased in vivo cleavage rates, has enabled us to express proinsulin, domain-D of staphylococcal protein A, hepcidin, interferon-alpha1, keratin-associated protein 10-4, green fluorescent protein, inhibitorial peptide of senescence-evasion-factor, monocyte chemoattractant protein-1 and toxic gyrase inhibitor, among others. This N(pro) expression system can be used as a generic tool for the high-level production of recombinant toxic peptides and proteins (up to 12 g/l) in Escherichia coli without the need for chemical or enzymatic removal of the fusion tag.
authors.affiliation: Austrian Center of Biopharmaceutical Technology, Muthgasse 18, A-1190 Vienna, Austria.
authors.forename: Clemens
authors.initials: C
authors.lastname: Achmüller
authors.forename: Waltraud
authors.initials: W
authors.lastname: Kaar
authors.forename: Karin
authors.initials: K
authors.lastname: Ahrer
authors.forename: Philipp
authors.initials: P
authors.lastname: Wechner
authors.forename: Rainer
authors.initials: R
authors.lastname: Hahn
authors.forename: Florian
authors.initials: F
authors.lastname: Werther
authors.forename: Hannes
authors.initials: H
authors.lastname: Schmidinger
authors.forename: Monika
authors.initials: M
authors.lastname: Cserjan-Puschmann
authors.forename: Franz
authors.initials: F
authors.lastname: Clementschitsch
authors.forename: Gerald
authors.initials: G
authors.lastname: Striedner
authors.forename: Karl
authors.initials: K
authors.lastname: Bayer
authors.forename: Alois
authors.initials: A
authors.lastname: Jungbauer
authors.forename: Bernhard
authors.initials: B
authors.lastname: Auer
mesh_terms: D001709:Biotechnology / Q000379:methods*; D006692:Classical Swine Fever Virus / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins