Comparative evaluation of two cell-free protein synthesis systems derived from Escherichia coli for genetic code reprogramming.

Genetic codes can be reprogrammed to code for non-proteinogenic amino acids during protein synthesis. Technologically, these non-proteinogenic amino acids are incorporated into proteins by artificially charging them to suppressor-tRNAs that can reprogram the existing codons. Several methods and systems for genetic code reprogramming have been reported including methods for charging non-proteinogenic amino acids to tRNA molecules, codons for reprogramming, and systems for protein synthesis. However, there has been no systematic, comparative evaluation of cell-free protein synthesis systems in genetic code reprogramming for their efficiencies and robustness even with their potential usefulness in the field. Here we compare two cell-free protein synthesis systems, the crude S12 and PURE system, with the codon systems, non-proteinogenic amino acids, and the positions in the protein for reprogramming as variables. We show that the combined use of CCCG four-nucleotide codon that is newly developed in this study and the crude S12 system is the most reliable and robust method of choice, while the use of traditional UAG amber stop codon along with an RNA aptamer toward peptide release factor 1 can yield the most plentiful product with certain variations.