Efficient In Vitro Full‐Sense‐Codons Protein Synthesis

Abstract

orthogonal suppressor tRNA holds the key to the incorporation efficiency. Recently, improved incorporation is achieved in E. coli strain C321.ΔA, in which all amber codons, accounting for 8% of stop codons in E. coli, [1] are replaced and prfA is removed away from the genome. [2] However, it will be extremely lethal for a cell to simultaneously interfere with all three stop codons. Saturation mutagenesis for molecular evolution is another application restricted by termination mechanisms. Stop codon involved in NNN randomized codon causes undesired premature termination. One stop codon is still generated even with NNK or NNS, moreover, the reduced codon set excludes codons with high usage frequency (Figure S1, Supporting Information). We anticipate that complete codon-dependent termination defect protein translation could efficiently liberate all codons for sense function and improve all the genetic code engineering applications (Figure 1).In the cell, termination of translation includes both the essential mechanism of class-I release factors and alternative mechanisms, [3] such as tmRNA (ssrA) mediated transtranslation [4] and translation stalling rescue. [5] As alternative termination ArfT in F. tularensis, [6] ResQ in Bacillus subtilis [7] are reported. In contrast to RF1, there is still no feasible approach for deletion of RF2, major class-I release factor in charge of the essential termination and plays a critical role in alternative terminations as well, i.e., post-peptidyl transfer quality control and alternative ribosome rescue. [8] However, cell viability becomes a barrier for in vivo assessment of global termination function. Therefore, there is strong curiosity about what if losing all essential termination machineries.Essential genes cannot be genetically deleted in a living cell. Pdt peptide containing 27 amino acids in length evolved from Mesoplasma florum Lon protease (mf-Lon) is demonstrated as being able to specifically target protein to efficient degradation in E. coli. [9] Pdt-tag is fused to nascent chain release factor RF1 and RRF respectively in E. coli MG1655pro, but failed on prfB encoding release factor RF2. Thus far, complete removal of all termination functions in either living cell or cell lysate was not yet achieved.Here, we present one efficient in vitro protein synthesis with 64 sense codons (iPSSC). The mf-Lon directed protein degradation is demonstrated as one efficient approach for the Termination of translation is essential but hinders applications of genetic code engineering, e.g., unnatural amino acids incorporation and codon randomization mediated saturation mutagenesis. Here, for the first time, it is demonstrated that E. coli Pth and ArfB together play an efficient translation termination without codon preference in the absence of class-I release factors. By degradation of the targeted protein, both essential and alternative termination types of machinery are completely removed to disable codon-dependent termination in cell extract. Moreover, a total of 153 engineered tRNAs...

Translation initiation consistency between in vivo and in vitro bacterial protein expression systems

“…The S30 cell extract was prepared as described in our previous study ( Wu et al, 2022 ), but we did not carry out the run-off reaction for high CFPS yields. Hence, the subpackage extract was directly frozen in liquid nitrogen and stored in the refrigerator at −80°C.…”

“…For the characterization of 5′-UTR in vitro , a standard CFPS reaction in the 20 µL reaction system was used containing 2 μL of PCR products and other reaction components same as described in a previous report ( Wu et al, 2022 ). The addition of 2 μL of the PCR products was optimal for achieving the highest yield in this CFPS reaction ( Supplementary Figure S4B ).…”

“…To facilitate the engineering of biological elements for cell-free systems, we tested the consistency of 5′-UTRs regulation in vivo and in vitro (Figure 4A). With the goal of constructing a robust, highyielding CFPS system, a reported protocol (Wu et al, 2022), was Frontiers in Bioengineering and Biotechnology frontiersin.org adopted to prepare S30-extracts derived from the cells of E. coli JM109 (DE3) and BL21 Star (DE3). To assess the potential ability of protein synthesis, we subsequently carried out the CFPS of the standard sfGFP template in a 20 μL batch reaction for 3 h at 30 °C.…”