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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.

SUBMITTER: Hohl A

PROVIDER: S-EPMC6700097 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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

Hohl Adrian A Karan Ram R Akal Anastassja A Renn Dominik D Liu Xuechao X Ghorpade Seema S Groll Michael M Rueping Magnus M Eppinger Jörg J

Scientific reports 20190819 1

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 ...[more]

PMID: 31427620

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