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

Handle URI:
http://hdl.handle.net/10754/626370
Title:
Engineering a promiscuous pyrrolysyl-tRNA synthetase by a high throughput FACS screen
Authors:
Hohl, Adrian; Karan, Ram; Akal, Anstassja; Renn, Dominik; Liu, Xuechao; Dharamarajnadar, Alaguraj; Ghoprade, Seema Arun ( 0000-0001-5576-1390 ) ; Groll, Michael; Rueping, Magnus ( 0000-0003-4580-5227 ) ; Eppinger, Jorg ( 0000-0001-7886-7059 )
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.
KAUST Department:
KAUST Catalysis Center (KCC); Physical Sciences and Engineering (PSE) Division
Citation:
Hohl 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.
Publisher:
Cold Spring Harbor Laboratory
Issue Date:
6-Dec-2017
DOI:
10.1101/229054
Type:
Preprint
Sponsors:
The 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.
Additional Links:
https://www.biorxiv.org/content/early/2017/12/05/229054
Appears in Collections:
Other/General Submission; Physical Sciences and Engineering (PSE) Division; KAUST Catalysis Center (KCC)

Full metadata record

DC FieldValue Language
dc.contributor.authorHohl, Adrianen
dc.contributor.authorKaran, Ramen
dc.contributor.authorAkal, Anstassjaen
dc.contributor.authorRenn, Dominiken
dc.contributor.authorLiu, Xuechaoen
dc.contributor.authorDharamarajnadar, Alagurajen
dc.contributor.authorGhoprade, Seema Arunen
dc.contributor.authorGroll, Michaelen
dc.contributor.authorRueping, Magnusen
dc.contributor.authorEppinger, Jorgen
dc.date.accessioned2017-12-14T12:34:04Z-
dc.date.available2017-12-14T12:34:04Z-
dc.date.issued2017-12-06en
dc.identifier.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.en
dc.identifier.doi10.1101/229054en
dc.identifier.urihttp://hdl.handle.net/10754/626370-
dc.description.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.en
dc.description.sponsorshipThe 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.en
dc.publisherCold Spring Harbor Laboratoryen
dc.relation.urlhttps://www.biorxiv.org/content/early/2017/12/05/229054en
dc.rightsArchived with thanks to bioRxiven
dc.subjectunnatural amino acidsen
dc.subjectamber suppressionen
dc.subjectpyrrolysyl-tRNA synthetaseen
dc.subjectnon-canonical amino acidsen
dc.subjectprotein engineeringen
dc.titleEngineering a promiscuous pyrrolysyl-tRNA synthetase by a high throughput FACS screenen
dc.typePreprinten
dc.contributor.departmentKAUST Catalysis Center (KCC)en
dc.contributor.departmentPhysical Sciences and Engineering (PSE) Divisionen
dc.eprint.versionPre-printen
dc.contributor.institutionTechnical University of Munich, Center for Integrated Protein Science Munich in the Department Chemistry, Garching, Germanyen
dc.contributor.institutionDepartment of Dyestuff Technology, Institute of Chemical Technology, Matunga, Mumbai, India 400019en
kaust.authorHohl, Adrianen
kaust.authorKaran, Ramen
kaust.authorAkal, Anstassjaen
kaust.authorRenn, Dominiken
kaust.authorLiu, Xuechaoen
kaust.authorDharamarajnadar, Alagurajen
kaust.authorGhoprade, Seema Arunen
kaust.authorRueping, Magnusen
kaust.authorEppinger, Jorgen
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