Cell-free co-production of an orthogonal transfer RNA activates efficient site-specific non-natural amino acid incorporation

TLDR

The authors present a cell‑free protein synthesis platform that simultaneously generates an orthogonal tRNA and a modified protein, enabling efficient, site‑specific incorporation of non‑natural amino acids and facilitating ribozyme screening. In the CFPS system, the orthogonal tRNA is transcribed from a linearized plasmid or crude PCR product and co‑produced with the target protein, with an evolved tRNA variant (o‑tRNA(opt)) improving Ef‑Tu recognition at high template concentrations. Using this method, soluble sfGFP containing p‑azido‑l‑phenylalanine or p‑propargyloxy‑l‑phenylalanine was produced at 0.9–1.7 mg ml⁻¹ (50–88 % suppression efficiency), with yields and suppression efficiencies largely independent of site and enhanced by the optimized tRNA.

Abstract

We describe a new cell-free protein synthesis (CFPS) method for site-specific incorporation of non-natural amino acids (nnAAs) into proteins in which the orthogonal tRNA (o-tRNA) and the modified protein (i.e. the protein containing the nnAA) are produced simultaneously. Using this method, 0.9-1.7 mg/ml of modified soluble super-folder green fluorescent protein (sfGFP) containing either p-azido-l-phenylalanine (pAzF) or p-propargyloxy-l-phenylalanine (pPaF) accumulated in the CFPS solutions; these yields correspond to 50-88% suppression efficiency. The o-tRNA can be transcribed either from a linearized plasmid or from a crude PCR product. Comparison of two different o-tRNAs suggests that the new platform is not limited by Ef-Tu recognition of the acylated o-tRNA at sufficiently high o-tRNA template concentrations. Analysis of nnAA incorporation across 12 different sites in sfGFP suggests that modified protein yields and suppression efficiencies (i.e. the position effect) do not correlate with any of the reported trends. Sites that were ineffectively suppressed with the original o-tRNA were better suppressed with an optimized o-tRNA (o-tRNA(opt)) that was evolved to be better recognized by Ef-Tu. This new platform can also be used to screen scissile ribozymes for improved catalysis.