Engineering a promiscuous pyrrolysyl-tRNA synthetase by a high throughput FACS screen
Gespers (Akal), Anastassja
Ghoprade, Seema Arun
KAUST DepartmentBiological & Organometallic Catalysis Laboratories
Biological and Environmental Sciences and Engineering (BESE) Division
Bioscience Core Lab
Cell Sorting and Cell Culture
Chemical Science Program
KAUST Catalysis Center (KCC)
NGS, qPCR and Single Cell Genomics
Office of the VP
Physical Science and Engineering (PSE) Division
Permanent link to this recordhttp://hdl.handle.net/10754/626370
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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