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

Adrian Hohl, Ram Karan, Anstassja Akal, Dominik Renn, Xuechao Liu, Alaguraj Dharamarajnadar, View ORCID ProfileSeema Ghoprade, Michael Groll, Magnus Rueping, View ORCID ProfileJörg Eppinger
doi: https://doi.org/10.1101/229054
Adrian Hohl
1King Abdullah University of Science and Technology (KAUST), KAUST Catalysis Center (KCC), Physical Sciences and Engineering Division (PSE), Thuwal, 23955-6900, Saudi Arabia
3Technical University of Munich, Center for Integrated Protein Science Munich in the Department Chemistry, Garching, Germany
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Ram Karan
1King Abdullah University of Science and Technology (KAUST), KAUST Catalysis Center (KCC), Physical Sciences and Engineering Division (PSE), Thuwal, 23955-6900, Saudi Arabia
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Anstassja Akal
1King Abdullah University of Science and Technology (KAUST), KAUST Catalysis Center (KCC), Physical Sciences and Engineering Division (PSE), Thuwal, 23955-6900, Saudi Arabia
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Dominik Renn
1King Abdullah University of Science and Technology (KAUST), KAUST Catalysis Center (KCC), Physical Sciences and Engineering Division (PSE), Thuwal, 23955-6900, Saudi Arabia
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Xuechao Liu
1King Abdullah University of Science and Technology (KAUST), KAUST Catalysis Center (KCC), Physical Sciences and Engineering Division (PSE), Thuwal, 23955-6900, Saudi Arabia
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Alaguraj Dharamarajnadar
1King Abdullah University of Science and Technology (KAUST), KAUST Catalysis Center (KCC), Physical Sciences and Engineering Division (PSE), Thuwal, 23955-6900, Saudi Arabia
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Seema Ghoprade
1King Abdullah University of Science and Technology (KAUST), KAUST Catalysis Center (KCC), Physical Sciences and Engineering Division (PSE), Thuwal, 23955-6900, Saudi Arabia
2Department of Dyestuff Technology, Institute of Chemical Technology, Matunga, Mumbai, India 400019
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  • ORCID record for Seema Ghoprade
Michael Groll
3Technical University of Munich, Center for Integrated Protein Science Munich in the Department Chemistry, Garching, Germany
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Magnus Rueping
1King Abdullah University of Science and Technology (KAUST), KAUST Catalysis Center (KCC), Physical Sciences and Engineering Division (PSE), Thuwal, 23955-6900, Saudi Arabia
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  • For correspondence: magnus.rueping@kaust.edu.sa jorg.eppinger@kaust.edu.sa
Jörg Eppinger
1King Abdullah University of Science and Technology (KAUST), KAUST Catalysis Center (KCC), Physical Sciences and Engineering Division (PSE), Thuwal, 23955-6900, Saudi Arabia
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  • ORCID record for Jörg Eppinger
  • For correspondence: magnus.rueping@kaust.edu.sa jorg.eppinger@kaust.edu.sa
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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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  • The authors declare no conflict of interest

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted December 05, 2017.
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Engineering a promiscuous pyrrolysyl-tRNA synthetase by a high throughput FACS screen
Adrian Hohl, Ram Karan, Anstassja Akal, Dominik Renn, Xuechao Liu, Alaguraj Dharamarajnadar, Seema Ghoprade, Michael Groll, Magnus Rueping, Jörg Eppinger
bioRxiv 229054; doi: https://doi.org/10.1101/229054
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Engineering a promiscuous pyrrolysyl-tRNA synthetase by a high throughput FACS screen
Adrian Hohl, Ram Karan, Anstassja Akal, Dominik Renn, Xuechao Liu, Alaguraj Dharamarajnadar, Seema Ghoprade, Michael Groll, Magnus Rueping, Jörg Eppinger
bioRxiv 229054; doi: https://doi.org/10.1101/229054

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