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A wide range of non-canonical amino acids (ncAAs) can be incorporated into proteins in living cells by using engineered aminoacyl-tRNA synthetase/tRNA pairs. However, most engineered tRNA synthetases are polyspecific; that is, they can recognize multiple rather than one ncAA. Polyspecificity of engineered tRNA synthetases imposes a limit to the use of genetic code expansion because it prevents specific incorporation of a desired ncAA when multiple ncAAs are present in the growth media. In this study, we employed directed evolution to improve substrate selectivity of polyspecific tRNA synthetases by developing substrate-selective readouts for flow-cytometry-based screening with simultaneous presence of multiple ncAAs. We applied this method to improve selectivity of two commonly used tRNA synthetases, p-cyano-L-phenylalanyl aminoacyl-tRNA synthetase (pCNFRS) and Nε-acetyl-lysyl aminoacyl-tRNA synthetase (AcKRS), with broad specificity. Evolved pCNFRS and AcKRS variants exhibit significantly improved selectivity for ncAAs p-azido-L-phenylalanine (pAzF) and m-iodo-L-phenylalanine (mIF), respectively. To demonstrate the utility of our approach, we used the newly evolved tRNA synthetase variant to produce highly pure proteins containing the ncAA mIF, in the presence of multiple ncAAs present in the growth media. In summary, our new approach opens up a new avenue for engineering next generation of tRNA synthetases with improved selectivity toward a desired ncAA.
This article was published in the following journal.
Name: ACS chemical biology
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A protein found in bacteria and eukaryotic mitochondria which delivers aminoacyl-tRNA's to the A site of the ribosome. The aminoacyl-tRNA is first bound to a complex of elongation factor Tu containing a molecule of bound GTP. The resulting complex is then bound to the 70S initiation complex. Simultaneously the GTP is hydrolyzed and a Tu-GDP complex is released from the 70S ribosome. The Tu-GTP complex is regenerated from the Tu-GDP complex by the Ts elongation factor and GTP. EC 3.6.1.-.
Peptide elongation factor 1 is a multisubunit protein that is responsible for the GTP-dependent binding of aminoacyl-tRNAs to eukaryotic ribosomes. The alpha subunit (EF-1alpha) binds aminoacyl-tRNA and transfers it to the ribosome in a process linked to GTP hydrolysis. The beta and delta subunits (EF-1beta, EF-1delta) are involved in exchanging GDP for GTP. The gamma subunit (EF-1gamma) is a structural component.
A subclass of enzymes that aminoacylate AMINO ACID-SPECIFIC TRANSFER RNA with their corresponding AMINO ACIDS.
Nonsusceptibility of bacteria to the action of TETRACYCLINE which inhibits aminoacyl-tRNA binding to the 30S ribosomal subunit during protein synthesis.
A dioxygenase and alkylation repair homolog that catalyzes the methylation of 5-carboxymethyl URIDINE to 5-methylcarboxymethyl uridine at the wobble position of the ANTICODON loop in TRANSFER RNA (tRNA) via its methyltransferase domain. It has a preference for tRNA (ARGININE) and tRNA (GLUTAMATE), and does not bind tRNA (LYSINE).