PROTEINS 2014 * Selective protein modifications at multiple sites in cell-free protein synthesis systems (#233)
Site specific modification of polypeptides with unnatural aminoacids (UAAs) is invaluable for exploring the details of their function and for endowing these molecules with novel functionalities. In particular, fluorescent spectroscopy that can provide information about protein structural dynamics relies on site-selective incorporation of two or more probes into a polypeptide chain. Presence of multiple reactive side chains in polypeptides precludes their site-selective labelling. Co-translational incorporation of UAAs is complicated by the fact that dyes larger than Bodipy FL are incorporated very inefficiently. This can be overcome by incorporating of UAAs that contain small bioorthogonal functionalities and their subsequent derivatization. The most common approach for incorporation of UAAs into polypeptide sequence is via codon orthogonal to translation machinery. These are either naturally occurring stop codons or artificially designed four-base codons. Although use of these methods both separately and in combination has been demonstrated, they suffer from severely compromised protein yields.
We developed an approach for effective protein modification via use of amber stop codon in combination with intein-mediated protein trans-splicing in cell-free protein synthesis systems. Here N-terminus of a protein could be effectively labelled with dye as a result of Ssp DnaB-mediated protein trans-splicing activity. Incorporation of UAA containing highly reactive bioorthogonal groups (cyclooctyne, trans-cyclooctene and bicycle[6.1.0]nonyne) is achieved by the use of recently developed M.mazei aminoacyl-tRNA transferase/tRNA pair. Importantly, use of universal translation enhancer (Species Independent Translational Sequence) allows us to label proteins in both prokaryotic E.coli and eukaryotic L.tarentolae extracts.
