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bioRxiv · 10.1101/229054

Engineering a promiscuous pyrrolysyl-tRNA synthetase by a high throughput FACS screen

Hohl, A.·Karan, R.·Akal, A.·Renn, D.·Liu, X.·Dharamarajnadar, A.·Ghoprade, S. A.·Groll, M.·Rueping, M.·Eppinger, J.
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Abstract

The 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.

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Hohl, A., Karan, R., Akal, A., Renn, D., Liu, X., Dharamarajnadar, A., Ghoprade, S. A., Groll, M., Rueping, M., Eppinger, J.. 2017-12-05. Engineering a promiscuous pyrrolysyl-tRNA synthetase by a high throughput FACS screen. https://doi.org/10.1101/229054

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