Increased gene translation stringency in mammalian cells by nonsense suppression at multiple permissive sites with a single noncanonical 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 noncanonical 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 noncanonical 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.