Site-Specific Chemoselective Pyrrolysine Analogues Incorporation Using the Cell-Free Protein Synthesis System

Cell-free protein synthesis (CFPS) is a fast and convenient way to synthesize proteins for analytical studies and applications. CFPS, when equipped with a suitable orthogonal pair, allows for protein-site-directed labeling with desired functionalities such as fluorescent dyes or therapeutic groups that are needed to tailor proteins for analytical applications. In this context, chemoselective reactive pyrrolysine analogues (CR-OAs) are of particular value, as this class of unnatural amino acids, among other useful properties, covers a wide range of different chemoselective reactions. In this study, we present a flexible approach that facilitates incorporation of CR-OAs in CFPS systems. In particular, a fairly simple addition of two expression plasmids in our cell-free system, one encoding pyrrolysyl-tRNA synthetase and the other one the target protein, enabled ribosomal synthesis of proteins in the half-milligram range with the pre-installed orthogonal reactivity, easily modifiable by using mild, copper-free bioorthogonal chemistry. Our CFPS system allows rapid and highly customizable expression, as shown by several examples of successful site-directed fluorescence labeling. The feasibility of our CFPS system for protein analytics is further proved by demonstrating the functional integrity of a labeled protein by interaction measurements using microscale thermophoresis.