Inducible genetic code expansion in eukaryotes.

Inducible genetic code expansion in eukaryotes.
Chembiochem. 2020 Jun 29;:
Authors: Koehler C, Estrada Girona G, Reinkemeier CD, Lemke EA
Abstract
Genetic code expansion (GCE) is a versatile tool to site-specifically incorporate a noncanonical amino acid (ncAA) into a protein to e.g. 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 efficiencies high levels of the orthogonal tRNA are required which could interfere with host cell functionality. To minimize adverse effects on the host, we developed an inducible GCE system, which enables us to switch on the tRNA or RS expression, when needed. In particular, we tested different promotors in 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 control of GCE components as well as efficiency. We find that only the T-REx system enables simultaneous control of tRNA and RS expression.
PMID: 32598534 [PubMed - as supplied by publisher]

Source: Chembiochem - June 28, 2020 Category: Biochemistry Authors: Koehler C, Estrada Girona G, Reinkemeier CD, Lemke EA Tags: Chembiochem Source Type: research