N(pro) fusion technology to produce proteins with authentic N termini in E. coli.
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.
Achmüller
Clemens
C
Austrian Center of Biopharmaceutical Technology, Muthgasse 18, A-1190 Vienna, Austria.
Kaar
Waltraud
W
Ahrer
Karin
K
Wechner
Philipp
P
Hahn
Rainer
R
Werther
Florian
F
Schmidinger
Hannes
H
Cserjan-Puschmann
Monika
M
Clementschitsch
Franz
F
Striedner
Gerald
G
Bayer
Karl
K
Jungbauer
Alois
A
Auer
Bernhard
B
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*
D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins