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Genetic code expansion enables the incorporation of unnatural amino acids into proteins thereby augmenting their physical and chemical properties. This is achieved by the reassignment of codons from their original sense to incorporate unnatural amino acids. The most commonly used methodology is stop codon suppression, which has resulted in numerous successful studies and applications in recent years. In these studies, many observations have been accumulated indicating that stop codon suppression efficiency depends on various cellular, operon and mRNA context effects. Predominant among these are mRNA context effects: the location of the stop codon along the mRNA governs, to a large extent, the efficiency and ability to successfully incorporate unnatural amino acids. Albeit their prevalence and importance, the mechanisms that govern context effects remain largely unknown. Herein, we will review what is known and yet to be understood with the intent to advance the propagation of genetic code expansion technology and to stimulate systematic research and debate of this open question., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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., (© 2016 Wiley Periodicals, Inc.)