Npro fusion technology: On-column complementation to improve efficiency in biopharmaceutical production

Npro fusion technology, a highly efficient system for overexpression of proteins and peptides in Escherichia coli, was further developed by splitting the autoprotease Npro 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 Npro Fusion Technology and thus allows more efficient production of recombinant proteins with reduced costs and in higher yields. Overall, the Npro 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.