Incorporation of nonstandard amino acids into proteins: principles and applications

The cellular ribosome shows a naturally evolved strong preference for the synthesis of proteins with standard amino acids. An in-depth understanding of the translation process enables scientists to go beyond this natural limitation and engineer translating systems capable of synthesizing proteins with artificially designed and synthesized non-standard amino acids (nsAA) featuring more bulky sidechains. The sidechains can be functional groups, with chosen biophysical or chemical activities, that enable the direct application of these proteins. Alternatively, the sidechains can be designed to contain highly reactive groups: enabling the ready formation of conjugates via a covalent bond between the sidechain and other chemicals or biomolecules. This co-translational incorporation of nsAAs into proteins allows for a vast number of possible applications. In this paper, we first systematically summarized the advances in the engineering of the translation system. Subsequently, we reviewed the extensive applications of these nsAA-containing proteins (after chemical modification) by discussing representative reports on how they can be utilized for different purposes. Finally, we discussed the direction of further studies which could be undertaken to improve the current technology utilized in incorporating nsAAs in order to use them to their full potential and improve accessibility across disciplines.