Npro 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.

Hybrid Modelling and Multi- Parametric Control of Bioprocesses

“…For instance, numerous studies show a higher matrix deposition or upregulation of relevant genes in comparison to static culture conditions when bone precursor cells are exposed to fluid shear stress [38][39][40][41][42][43]. Although several tailor-made [10,11,44] and commercially available [45][46][47] perfusion systems were developed and successfully used, there is a lack of automated sensor-controlled systems that allow the cultivation of multiple independent replicates under different conditions.…”

An optimized Npro-based method for the expression and purification of intrinsically disordered proteins for an NMR study

“…21 This His x6 -tagged N pro gene was ligated downstream of the T7 promoter of pET-21b between NdeI and BamHI sites. A SpeI site preceding the autoproteolytic cleavage site Cys168 and a short linker containing several restriction sites were engineered as a multi-cloning site, resulting in the pET-N pro vector (Supplementary Fig.…”

“…The N pro fusion technology was originally developed by Auer et al for producing recombinant proteins with native amino-terminal amino acids. 21 The N-terminal autoprotease N pro from classical swine fever virus was selected as a fusion partner. The N pro (EDDIE) mutant was further developed.…”

“…The N pro (EDDIE) mutant was further developed. 21 The fusion protein of N pro (EDDIE) and the protein of interest is first deposited in bacterial inclusion bodies and then refolded. During the refolding process, N pro (EDDIE) recovers its autoproteolytic activity to cleave its own C-terminus, thereby releasing the protein of interest with its native N-terminal amino acid.…”

Refolding of Npro fusion proteins

“…This system is based on the autoprotease N pro derived from classical swine fever virus or its mutant EDDIE as an Nterminal self-cleavable fusion tag (Achmüller et al, 2007). N pro exhibits the unique feature of autoproteolytic activity at its own C-terminus (Rümenapf et al, 1998;Stark et al, 1993).…”