Adding ‘splice ’ to protein engineering

The field of protein engineering has always attracted a multidisciplinary cast of characters ranging from biologists to organic chemists. Consequently, a variety of different protein engineering techniques have been described which encompass the purely synthetic and the purely biosynthetic. Of these, heterologous expression of recombinantly cloned genes is by far the most commonly employed route to engineered proteins — in part because the approach is readily accessible to most researchers. Although site-directed mutagenesis is facilitated by the ‘replicative fidelity and efficiency of living organisms ’ [1], this of course also constrains the approach to the naturally occurring amino acids. This limitation is largely overcome by elegant chemistry-driven approaches, such as unnatural amino acid mutagenesis [2] and protein total or semi-synthesis via the chemical [3–9] or enzymatic [10–13] ligation of unprotected peptides. These approaches are, however, technically demanding, a feature which has prevented their widespread use by the biological community. Recently, a novel protein semi-synthesis approach was reported [14–16] that harnesses the phenomenon of protein splicing (for review see [17]), and in the process renders proteins harboring unnatural building blocks accessible to most researchers. This short review describes the genesis of this methodology, terme