4.3 Article

Efficient In Vitro Full-Sense-Codons Protein Synthesis

Journal

ADVANCED BIOLOGY
Volume 6, Issue 10, Pages -

Publisher

WILEY-V C H VERLAG GMBH
DOI: 10.1002/adbi.202200023

Keywords

codon reassignment; protein degradation; synthetic biology; translation termination

Funding

  1. National Key Research and Development Program of China [2019YFA0904103]
  2. National Natural Science Foundation of China [21778039, 21621004]

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This study demonstrates that the E. coli Pth and ArfB proteins can efficiently terminate translation without codon preference in the absence of class-I release factors. By degrading the target protein, both essential and alternative termination machinery types are removed, disabling codon-dependent termination in cell extract. The researchers also screened 153 engineered tRNAs to construct a codon-dependent termination defect in vitro protein synthesis system, called iPSSC, which efficiently decodes all stop codons. The full sense genetic code achieved significant improvement in incorporating unnatural amino acids and synthesizing proteins with consecutive NNN codons, suggesting great potential for building artificial protein synthesis beyond the cell.
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 are screened for efficient all stop-codons decoding to construct a codon-dependent termination defect in vitro protein synthesis with all 64 sense-codons, iPSSC. Finally, this full sense genetic code achieves significant improvement in the incorporation of distinct unnatural amino acids at up to 12 positions and synthesis of protein encoding consecutive NNN codons. By decoding all information in nucleotides to amino acids, iPSSC may hold great potential in building artificial protein synthesis beyond the cell.

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