To assist in the characterization of protein complexes we have developed a modular approach to fusion protein generation that relies upon Sortase-mediated and Native chemical ligation using synthetic Peptide linkers (SNaPe)1 to link separately expressed proteins. This technique makes use of two separate linking steps – sortase-mediated and native chemical ligation – combined with various synthetic peptide linkers to generate libraries of fusion proteins. We have demonstrated that SNaPe can make fusion protein constructs of biosynthetic enzymes from aglycone assembly during glycopeptide antibiotic biosynthesis, including fusion proteins normally inaccessible via direct expression of the fusion construct itself. This demonstrates potential advantages of SNaPe to not only access fusion proteins that have been previously unavailable for biochemical and structural characterization but also to do so in a manner that enables the linker itself to be controlled during fusion protein generation.
1. Ulrich V, Cryle MJ. J Pept Sci. 2017 23(1):16-27.