Non-Standard Amino Acid Incorporation Using a Ribosome with Tethered Subunits

Previously, we developed an orthogonal ribosome with tethered subunits (oRibo-T) capable of translating specific orthogonal messenger RNAs (o-mRNA). oRibo-T allows researchers to investigate and engineer the ribosome’s 50S subunit in vivo without perturbing cell viability. The ribosome’s 50S subunit contains the peptidyltransferase center (PTC), which catalyzes peptide bond formation, and the exit tunnel, through which the nascent polypeptide is excreted. By decoupling the catalytic and secretion functions of the orthogonal ribosome from the pool of wild type ribosomes, oRibo-T represents a significant advance to previous works developing in vivo orthogonal translation machinery. Orthogonal translation systems have been applied in synthetic biology to incorporate non-standard amino acids (nsAAs) into proteins. Site-specific incorporation of nsAAs into proteins via amber suppression, where an nsAA is incorporated at a site corresponding to the amber codon (UAG) in the mRNA, has expanded our capacity for producing a variety of proteins for applications such as studying protein structure and function, mimicking posttranslational modifications, and producing improved protein therapeutics. Here, we apply oRibo-T for site-specific incorporation of nsAAs into proteins towards evolving oRibo-T’s 50S subunit for incorporating nsAAs currently limited by the architecture of the wild type PTC. By combining oRibo-T with existing orthogonal translation systems developed for synthetic biology, we showcase its applicability and potential for future efforts in engineering the translation machinery for both engineering and fundamental science applications.