Engineering a Polyspecific Pyrrolysyl-tRNA Synthetase by a High Throughput FACS Screen.

Adrian Hohl; Ram Karan; Anastassja Gespers (Akal); Dominik Renn; Xuechao Liu; Seema Arun Ghoprade; Michael Groll; Magnus Rueping; Jörg Eppinger

doi:10.1038/s41598-019-48357-0

Engineering a Polyspecific Pyrrolysyl-tRNA Synthetase by a High Throughput FACS Screen.

Adrian Hohl, Ram Karan, Anastassja Gespers (Akal), Dominik Renn, Xuechao Liu, Seema Arun Ghoprade, Michael Groll, Magnus Rueping, Jörg Eppinger

Research output: Contribution to journal › Article › peer-review

26 Scopus citations

Abstract

The Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNAPyl are extensively used to add non-canonical amino acids (ncAAs) to the genetic code of bacterial and eukaryotic cells. However, new ncAAs often require a cumbersome de novo engineering process to generate an appropriate PylRS/tRNAPyl pair. We here report a strategy to predict a PylRS variant with novel properties. The designed polyspecific PylRS variant HpRS catalyzes the aminoacylation of 31 structurally diverse ncAAs bearing clickable, fluorinated, fluorescent, and for the first time biotinylated entities. Moreover, we demonstrated a site-specific and copper-free conjugation strategy of a nanobody by the incorporation of biotin. The design of polyspecific PylRS variants offers an attractive alternative to existing screening approaches and provides insights into the complex PylRS-substrate interactions.

Original language	English (US)
Journal	Scientific reports
Volume	9
Issue number	1
DOIs	https://doi.org/10.1038/s41598-019-48357-0
State	Published - Aug 19 2019

Bibliographical note

KAUST Repository Item: Exported on 2020-10-01
Acknowledgements: Te 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 fnancial support. We are grateful to Prof. Peter G. Schultz (Te Scripps Research Institute, La Jolla, CA) for kindly providing the original pEVOL-PylRS plasmid.

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@article{f86d13c4ae764cc8aef78a73600881cb,

title = "Engineering a Polyspecific Pyrrolysyl-tRNA Synthetase by a High Throughput FACS Screen.",

abstract = "The Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNAPyl are extensively used to add non-canonical amino acids (ncAAs) to the genetic code of bacterial and eukaryotic cells. However, new ncAAs often require a cumbersome de novo engineering process to generate an appropriate PylRS/tRNAPyl pair. We here report a strategy to predict a PylRS variant with novel properties. The designed polyspecific PylRS variant HpRS catalyzes the aminoacylation of 31 structurally diverse ncAAs bearing clickable, fluorinated, fluorescent, and for the first time biotinylated entities. Moreover, we demonstrated a site-specific and copper-free conjugation strategy of a nanobody by the incorporation of biotin. The design of polyspecific PylRS variants offers an attractive alternative to existing screening approaches and provides insights into the complex PylRS-substrate interactions.",

author = "Adrian Hohl and Ram Karan and {Gespers (Akal)}, Anastassja and Dominik Renn and Xuechao Liu and Ghoprade, {Seema Arun} and Michael Groll and Magnus Rueping and J{\"o}rg Eppinger",

note = "KAUST Repository Item: Exported on 2020-10-01 Acknowledgements: Te 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 fnancial support. We are grateful to Prof. Peter G. Schultz (Te Scripps Research Institute, La Jolla, CA) for kindly providing the original pEVOL-PylRS plasmid.",

year = "2019",

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doi = "10.1038/s41598-019-48357-0",

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AU - Karan, Ram

AU - Gespers (Akal), Anastassja

AU - Renn, Dominik

AU - Liu, Xuechao

AU - Ghoprade, Seema Arun

AU - Groll, Michael

AU - Rueping, Magnus

AU - Eppinger, Jörg

N1 - KAUST Repository Item: Exported on 2020-10-01 Acknowledgements: Te 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 fnancial support. We are grateful to Prof. Peter G. Schultz (Te Scripps Research Institute, La Jolla, CA) for kindly providing the original pEVOL-PylRS plasmid.

PY - 2019/8/19

Y1 - 2019/8/19

N2 - The Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNAPyl are extensively used to add non-canonical amino acids (ncAAs) to the genetic code of bacterial and eukaryotic cells. However, new ncAAs often require a cumbersome de novo engineering process to generate an appropriate PylRS/tRNAPyl pair. We here report a strategy to predict a PylRS variant with novel properties. The designed polyspecific PylRS variant HpRS catalyzes the aminoacylation of 31 structurally diverse ncAAs bearing clickable, fluorinated, fluorescent, and for the first time biotinylated entities. Moreover, we demonstrated a site-specific and copper-free conjugation strategy of a nanobody by the incorporation of biotin. The design of polyspecific PylRS variants offers an attractive alternative to existing screening approaches and provides insights into the complex PylRS-substrate interactions.

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Engineering a Polyspecific Pyrrolysyl-tRNA Synthetase by a High Throughput FACS Screen.

Abstract

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