Engineering elongation factor Tu and tRNAs to better accommodate non-canonical amino acids during translation

EF-Tu;Non-canonical amino acid;Orthogonal translation system;Genetic code expansion;Polyspecificity;Protein engineering;Ribosomal translation;Elongation factors

DeLey Cox, Vanessa E. ; Gaucher, Eric A. Williams, Loren D. Finn, M. G. Chemistry and Biochemistry Oyelere, Adegboyega K. Hud, Nicholas V. ; Gaucher, Eric A.

University:Georgia Institute of Technology

Department:Chemistry and Biochemistry

关键词: EF-Tu;    Non-canonical amino acid;    Orthogonal translation system;    Genetic code expansion;    Polyspecificity;    Protein engineering;    Ribosomal translation;    Elongation factors;   

Others  :  https://smartech.gatech.edu/bitstream/1853/63483/1/DELEYCOX-DISSERTATION-2018.pdf

美国|英语

来源: SMARTech Repository

【 摘 要 】

Non-canonical amino acid (ncAA) incorporation has led to significant advances in protein science and engineering. Traditionally, incorporation of ncAAs is achieved via amber codon suppression using an engineered orthogonal aminoacyl-tRNA synthetase:tRNA pair. However, as more complex protein products are targeted, researchers are identifying additional barriers limiting the scope of currently available ncAA systems. One barrier is elongation factor Tu (EF-Tu), a protein responsible for proof-reading aa-tRNAs that substantially restricts ncAA scope by limiting ncaa-tRNA delivery to the ribosome. Researchers have responded by engineering ncAA-compatible EF-Tus for key ncAAs. However, this approach fails to address the extent to which EF-Tu inhibits efficient ncAA incorporation. Herein, we describe our efforts to transform how EF-Tu is utilized to incorporate ncAAs. Leveraging computational methods, we present data on four EF-Tu libraries and two tRNA libraries used in both in vitro and in vivo translation. Ultimately, the in vivo assay led to the identification of EF-Tu variants which accept non-native substrates. By mass spectrometry, we demonstrate two variants have expanded substrate acceptance. Fitness tests show a polyspecific EF-Tu is an accepted addition to the translation machinery and improves host organism fitness relative to an ncAA-specific engineered EF-Tu. These results lend credence to our choice of computational method (in this case REAP) and also suggest that EF-Tu and SelB may have a shared, substrate-promiscuous ancestor. Overall, we believe this approach complements current research highlighting the advantages of improved OTSs and promotes a more comprehensive approach critical to achieving future goals.