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

Adrian Hohl, Ram Karan, Anastassja Akal, Dominik Renn, Xuechao Liu, Seema Ghorpade, Michael Groll, Magnus Rueping, Jörg Eppinger

Research output: Contribution to journalArticlepeer-review

22 Scopus citations


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 languageEnglish
Article number11971
JournalScientific Reports
Issue number1
StatePublished - 1 Dec 2019


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