Recombinant Optimization of an Orthogonal Tryptophanyl

Suppressor tRNA

Pothukuchy A, Ellefson JW, Ellington AD, Hughes RA*
The engineering of novel synthetic systems and components will lead to the creation of novel biologically derived or inspired products ranging from commodity chemicals to synthetic proteins. For the creation of synthetically augmented proteins it is possible to further expand the diversity of the genetic code beyond 20 amino acids with additional synthetic amino acids incorporation at specific sites in proteins using orthogonal aminoacyl-tRNA synthetase/tRNA pairs. We have previously shown that the tryptophanyl tRNA synthetase/tRNA pair is an attractive candidate for achieving the goal of unnatural amino acid incorporation because of its spacious binding pocket and its potential to accommodate large and bulky synthetic amino acids. Here we report the development of the next generation of orthogonal tryptophanyl-tRNAs that are not only superior in their suppression ability but are also truly orthogonal in E.coli system. Rational design was used to create novel tRNA variants by making modifications on the acceptor arm as well as on the anticodon stem region of the parental tRNA sequences. These next generation tRNAs are not only useful for improved incorporation of unnatural amino acids into proteins but are also useful for understanding the capacity of novel orthogonal translation system components for applications in synthetic biology.

References:

1. Ellefson, J.W., Meyer, A.J., Hughes, R.A., Cannon, J. R., Brodbelt, J.S. and Ellington, A.D. (2013). Directed evolution of genetic parts and circuits by compartmentalized partnered replication. Nat Biotechnol., 32(1): 97-101.
2. Hughes, R..A. and Ellington A.D. (2010).Rational design of an orthogonal tryptophanyl nonsense suppressor tRNA. Nucleic Acids Res., 38(19): 6813-30.
3. Chatterjee, A., Xiao, H., Yang, P.Y., Soundararajan, G. and Schultz, P.G. (2013).A tryptophanyl-tRNA synthetase/tRNA pair for unnatural amino acid mutagenesis in E. coli. Angew Chem Int Ed Engl., 52(19): 5106-9.