Optimisation of chemical protein cleavage for erythropoietin semi-synthesis using native chemical ligation†

Chemical ligation is a popular and rapidly expanding area of research impacting numerous aspects of organic chemistry, biomolecular and materials science. Native chemical ligation (NCL) in particular, has found numerous applications in such areas. This is likely because NCL is a chemoselective coupling reaction between two, normally peptide, components, one bearing a thioester and the other an N-terminal cysteine functionality that reliably takes place in aqueous solution and in the absence of protecting groups. Additionally NCL has interfaced itself completely with biology in that the required thioester and cysteine bearing components can be obtained through chemical synthesis or genetic manipulation making NCL widely accessible to chemists and biologists alike. We reported the first use of CNBr-cleaved protein fragments for production of N-terminal cysteine containing components used in native chemical ligation from N-terminally His10tagged precursors (Fig. 1a). Expression of proteins in bacteria with an N-terminal cysteine residue is not a trivial procedure. While some proteins may be amenable to direct expression of a free N-terminal cys residue, more commonly a pre-cysteine sequence, which relies on the availability of a selective protease to remove this sequence and unmask the free cysteine prior to NCL, is employed. In our case we investigated the CNBr cleavage reaction because we could neither express the protein directly with an N-terminal cysteine nor cleave a pre-cysteine sequence enzymatically owing to the insolubility of the protein fragment. This is not an uncommon phenomenon, indeed it is remarkable that so many bacterially expressed protein fragments, that are often the reagents for NCL, are soluble to begin with owing to their lack of a complete primary structure. The fact that our protein fragments were soluble only in denaturing reagents or high concentrations of detergent made them incompatible with commonly employed proteases (e.g. Factor Xa). Herein we describe the optimisation of the CNBr cleavage protocol highlighting important features of the process and demonstrate how proteins that have been subjected to this treatment retain