TY - JOUR
T1 - Engineering a Polyspecific Pyrrolysyl-tRNA Synthetase by a High Throughput FACS Screen.
AU - Hohl, Adrian
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.
AB - 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.
UR - http://hdl.handle.net/10754/656577
UR - http://www.nature.com/articles/s41598-019-48357-0
UR - http://www.scopus.com/inward/record.url?scp=85070835482&partnerID=8YFLogxK
U2 - 10.1038/s41598-019-48357-0
DO - 10.1038/s41598-019-48357-0
M3 - Article
C2 - 31427620
SN - 2045-2322
VL - 9
JO - Scientific reports
JF - Scientific reports
IS - 1
ER -