Selective and Site-Specific Incorporation of Nonstandard Amino Acids Within Proteins for Therapeutic Applications

The incorporation of nonstandard amino acids (nsAAs) within protein sequences has broadened the chemical functionalities available for use in the study, prevention, or treatment of disease. The ability to genetically encode the introduction of nsAAs at precise sites of target recombinant proteins has enabled numerous applications such as bioorthogonal conjugation, thrombin inhibition, intrinsic biological containment of live organisms, and immunochemical termination of self-tolerance. Genetic systems that perform critical steps in enabling nsAA incorporation are known as orthogonal translation systems or orthogonal aminoacyl-tRNA synthetase/tRNA pairs. In Escherichia coli, several of these have been designed to accept novel nsAAs. Certain endogenous proteins, codon context, and standard amino acid concentrations can affect the yield of recombinant protein, the rate of nsAA incorporation within off-target proteins, and the rate of misincorporation due to near-cognate suppression or misacylation of orthogonal tRNA with standard amino acids. As a result, a significant body of work has been performed in engineering the E. coli genome to alleviate these issues. Here, we describe common methods applicable to nsAA incorporation within proteins in E. coli for sufficient purity and characterization for downstream therapeutic applications.