Increased gene translation stringency in mammalian cells by nonsense suppression at multiple permissive sites with a single non-canonical amino acid.

: The considerable potential of engineered cells compels the development of strategies for the stringent control of gene expression. A promising approach is the introduction of a premature stop codon (PTC) into a selected gene that is expressed only in the presence of non-canonical amino acids through nonsense suppression. Here, different strategies of amber PTC readthrough in mammalian cells were tested. The use of a tRNA synthetase together with a TAG codon-specific tRNA achieved PTC readthrough depending on the addition of a non-canonical amino acid (4-benzoyl-L-phenylalanine; Bpa). While single TAG codon incorporation exhibited detectable expression of the reporter protein even in the absence of Bpa, the use of a double PTC enabled virtually leakage-free functional gene translation. The introduction of an additional 5'-PTC, therefore, represents a generally applicable strategy to increase stringency in gene translation.