In vitro suppression of two different stop codons

ABSTRACT Proteins play a crucial role in all living organisms, with the 20 natural amino acids as their building blocks. Unnatural amino acids are synthetic derivatives of these natural building blocks. These amino acids have unique chemical or physical properties as a result of their specific side chain residues. Their incorporation into proteins through ribosomal translation in response to one of the stop codons has opened a new way to manipulate and study proteins by enabling new functionalities, thus expending the genetic code. Different unnatural amino acids have different functionalities, hence, the ability to incorporate two different unnatural amino acids, in response to two different stop codons into one protein is a useful tool in protein manipulation. This ability has been achieved previously only in in vivo translational systems, however, with limited functionality. Herein, we report the incorporation of two different unnatural amino acids in response to two different stop codons into one protein, utilizing a cell‐free protein synthesis system. Biotechnol. Bioeng. 2017;114: 1065–1073. © 2016 Wiley Periodicals, Inc. A protein containing two different unnatural amino acids, incorporated in response to two different stop‐codons, was generated through a combination of two different cell‐free extracts. Each extract contained a different tRNA‐synthetase/tRNA pair prior to lysis, thus resulting in two lysates with the ability to incorporate a different unnatural amino acid. The authors were able to produce a protein containing two biorthogonal chemical‐handles, which were tethered to fluorophores, and susequently was used for FRET.