Towards Improved Applications of Cell-Free Protein Biosynthesis - The Influence of mRNA Structure and Suppressor tRNAS on the Efficiency of the System

Abstract

The cell-free protein biosynthesis has the potential to become a powerful technology for the biochemical research in particular in the determination of the structure and function of proteins. The number of possible applications is rising with the obtainable yields and with the expanded feasibility of introducing modified amino acids into proteins. Here we describe the influence of two RNA translation components, the mRNA and the suppressor tRNA, on the efficiency of protein biosynthesis. It is shown that the rate limiting factor of the cell-free translation of the two proteins dihydrofolate reductase (DHFR) and fatty acid binding protein (FABP) is not the initiation or termination step. The efficiency of peptide bond formation in the nascent protein varies between the two genes but is independent on the size of the coding sequences. The poor translation of DHFR can be improved when its coding sequence is fused with a part of the more efficiently translated FABP gene. We compared different amber suppressor tRNAs on the level of translational efficiency and aminoacylation capacity, Our results show that in most cases the aminoacylation rate of the tRNAs is not the limiting factor of suppression. An E. coli tRNALeu(CUA) exhibits the highest translational efficiency of the examined tRNAs, So this tRNALeu(CUA), may be a starting point to construct more efficient tRNAs for the introduction of unnatural amino acids into proteins in the in vitro translation system by eliminating the synthetase mediated aminoacylation of the tRNA.