title: N(pro) fusion technology: On-column complementation to improve efficiency in biopharmaceutical production.
abstract: N(pro) fusion technology, a highly efficient system for overexpression of proteins and peptides in Escherichia coli, was further developed by splitting the autoprotease N(pro) into two fragments to generate a functional complementation system. The size of the expression tag is thus reduced from 168 to 58 amino acids, so by 66%. Upon complementation of the fragments auto-proteolytic activity is restored. This process has been shown for three model proteins of different size, a short 16 aa-peptide, MCP-1, and lysozyme. Moreover, the complementation was still functional after immobilization of the N-terminal fragment to a solid support which enables recycling of the immobilized fragment. This strategy enhances overall productivity of N(pro) Fusion Technology and thus allows more efficient production of recombinant proteins with reduced costs and in higher yields. Overall, the N(pro) complementation system has, depending on the size of the target molecule, potential to increase the productivity up to 4 fold for batch refolding and even more for on-column refolding strategies by the proven possibility of regeneration of the immobilized fragment.
authors.affiliation: Austrian Centre of Industrial Biotechnology, Muthgasse 11, 1190 Vienna, Austria.
authors.forename: S
authors.initials: S
authors.lastname: Schindler
authors.affiliation: Austrian Centre of Industrial Biotechnology, Muthgasse 11, 1190 Vienna, Austria.
authors.forename: B
authors.initials: B
authors.lastname: Missbichler
authors.affiliation: Austrian Centre of Industrial Biotechnology, Muthgasse 11, 1190 Vienna, Austria; Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Muthgasse 18, 1190 Vienna, Austria.
authors.forename: C
authors.initials: C
authors.lastname: Walther
authors.affiliation: Austrian Centre of Industrial Biotechnology, Muthgasse 11, 1190 Vienna, Austria.
authors.forename: M
authors.initials: M
authors.lastname: Sponring
authors.affiliation: Austrian Centre of Industrial Biotechnology, Muthgasse 11, 1190 Vienna, Austria; Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Muthgasse 18, 1190 Vienna, Austria.
authors.forename: M
authors.initials: M
authors.lastname: Cserjan-Puschmann
authors.affiliation: Institute of Biochemistry, University of Innsbruck, Innrain 82, 6020 Innsbruck, Austria.
authors.forename: B
authors.initials: B
authors.lastname: Auer
authors.affiliation: Austrian Centre of Industrial Biotechnology, Muthgasse 11, 1190 Vienna, Austria; Institute of Biochemistry, University of Innsbruck, Innrain 82, 6020 Innsbruck, Austria.
authors.forename: R
authors.initials: R
authors.lastname: Schneider
authors.affiliation: Austrian Centre of Industrial Biotechnology, Muthgasse 11, 1190 Vienna, Austria; Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Muthgasse 18, 1190 Vienna, Austria. Electronic address: astrid.duerauer@boku.ac.at.
authors.forename: A
authors.initials: A
authors.lastname: Dürauer
mesh_terms: D000595:Amino Acid Sequence; D018932:Chemokine CCL2 / Q000302:isolation & purification / Q000378:metabolism; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009113:Muramidase / Q000302:isolation & purification / Q000378:metabolism; D010447:Peptide Hydrolases; D010455:Peptides / Q000302:isolation & purification / Q000378:metabolism; D058849:Protein Refolding; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification* / Q000378:metabolism; D015854:Up-Regulation
chemical_list: C111835:CCL2 protein, human; D018932:Chemokine CCL2; D010455:Peptides; D011993:Recombinant Fusion Proteins; D009113:Muramidase; D010447:Peptide Hydrolases
keywords: Autoprotease N(pro); Immobilization; Intein; Protein fragment complementation; Recombinant protein production