PT  - JOURNAL ARTICLE
AU  - Adrian Hohl
AU  - Ram Karan
AU  - Anstassja Akal
AU  - Dominik Renn
AU  - Xuechao Liu
AU  - Alaguraj Dharamarajnadar
AU  - Seema Ghoprade
AU  - Michael Groll
AU  - Magnus Rueping
AU  - Jörg Eppinger
TI  - Engineering a promiscuous pyrrolysyl-tRNA synthetase by a high throughput FACS screen
AID  - 10.1101/229054
DP  - 2017 Jan 01
TA  - bioRxiv
PG  - 229054
4099  - http://biorxiv.org/content/early/2017/12/05/229054.short
4100  - http://biorxiv.org/content/early/2017/12/05/229054.full
AB  - 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.