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

摘要

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 SI 2 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 SI 2 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.