Engineering a promiscuous pyrrolysyl-tRNA synthetase by a high throughput FACS screen
Ghoprade, Seema Arun
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AbstractThe Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNAPyl are used to facilitate the incorporation of non-canonical amino acids (ncAAs) into the genetic code of bacterial and eukaryotic cells by orthogonally reassigning the amber codon. Currently, the incorporation of new ncAAs requires a cumbersome engineering process composed of several positive and negative selection rounds to select the appropriate PylRS/tRNAPyl pair. Our fast and sensitive engineering approach required only a single FACS selection round to identify 110 orthogonal PylRS variants for the aminoacylation of 20 ncAAs. Pocket-substrate relationship from these variants led to the design of a highly promiscuous PylRS (HpRS), which catalyzed the aminoacylation of 31 structurally diverse lysine derivatives bearing clickable, fluorinated, fluorescent, and biotinylated entities. The high speed and sensitivity of our approach provides a competitive alternative to existing screening methodologies, and delivers insights into the complex PylRS-substrate interactions to facilitate the generation of additional promiscuous variants.
CitationHohl A, Karan R, Akal A, Renn D, Liu X, et al. (2017) Engineering a promiscuous pyrrolysyl-tRNA synthetase by a high throughput FACS screen. Available: http://dx.doi.org/10.1101/229054.
SponsorsThe research reported in this publication was supported by funding from King Abdullah University of Science and Technology (KAUST). We thank the SFB749/A10 (M.G.) for financial support. We are grateful to Prof. Peter G. Schultz (The Scripps Research Institute, La Jolla, CA) for kindly providing the original pEVOL-PylRS plasmid.
PublisherCold Spring Harbor Laboratory