6533b833fe1ef96bd129c1c1

RESEARCH PRODUCT

Inducible Genetic Code Expansion in Eukaryotes

Christine KoehlerChristopher D. ReinkemeierGemma Estrada GironaEdward A. Lemke

subject

Context (language use)Computational biology010402 general chemistry01 natural sciencesBiochemistryInsert (molecular biology)Amino Acyl-tRNA SynthetasesRNA TransferEscherichia coliHumansunnatural amino acidAmino AcidsMolecular BiologyT-RExchemistry.chemical_classificationTet-On010405 organic chemistryChemistryCommunicationOrganic ChemistryEukaryotaGenetic codeamber suppressionCommunications0104 chemical sciencesAmino acidMaximum efficiencyFluorescent labellingHEK293 CellsGenetic CodePylRSTransfer RNAMolecular MedicineAmber Stop Codon

description

Abstract Genetic code expansion (GCE) is a versatile tool to site‐specifically incorporate a noncanonical amino acid (ncAA) into a protein, for example, to perform fluorescent labeling inside living cells. To this end, an orthogonal aminoacyl‐tRNA‐synthetase/tRNA (RS/tRNA) pair is used to insert the ncAA in response to an amber stop codon in the protein of interest. One of the drawbacks of this system is that, in order to achieve maximum efficiency, high levels of the orthogonal tRNA are required, and this could interfere with host cell functionality. To minimize the adverse effects on the host, we have developed an inducible GCE system that enables us to switch on tRNA or RS expression when needed. In particular, we tested different promotors in the context of the T‐REx or Tet‐On systems to control expression of the desired orthogonal tRNA and/or RS. We discuss our result with respect to the control of GCE components as well as efficiency. We found that only the T‐REx system enables simultaneous control of tRNA and RS expression.

yearjournalcountryeditionlanguage
2020-05-28ChemBioChem
https://doi.org/10.1002/cbic.202000338