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.