2.6. Incorporation of unnatural amino acids into proteins

In this study, we used two different systems that expand the genetic code of a human cell and allow the incorporation of either trans-cyclooctene L-lysine (TCO*A) or p-Azido-L-phenylalanine (AzF) into eGFP [26]. TCO incorporation was mediated by an orthogonal tRNA/ aaRS pair from Methanosarcina mazei co-encoded on a single plasmid (plasmid was supplied by Edward Lemke, EMBL Heidelberg, Germany) [27]. Incorporation of AzF is based on the orthogonal tRNA^Tyr_CUA (tRNA fusion construct from H. sapiens and Bacillus stearothermophilus) and the E. coli aaRS encoded on two separate plasmids (plasmid was supplied by T. Huber, Rockefeller University New York, USA) [28]. For transfection of human cells, conditions that were introduced above were used. In addition to the plasmids that encode the respective orthogonal tRNA and aaRS pair, the eGFP^T39Amber expression vector was transfected. The total amount of plasmid DNA and transfection reagent was identical to the procedures described above.

TCO*A and AzF were obtained from SiChem (Bremen, Germany) and ChemImpex International (Wood Dale, USA), respectively. The uaas were dissolved in 200 mM NaOH and 15% DMSO to yield a 100 mM stock solution that can be stored at − 20 °C. At the day of transfection, a working solution of 25 mM uaa was generated by a 1:4 dilution with 1 M Hepes/KOH, pH 7.5. Prior transfection, the medium was exchanged to freshly prepared growth medium, containing the respective uaa in the desired concentration (50 µM were already sufficient for optimal incorporation of each uaa).

The efficiency of the uaa incorporation was monitored by fluorescence microscopy of living cells (Zeiss Axiovert 35 microscope connected to a Color view soft imaging system). For acquisition of fluorescence-images a filter-block (λ_ex = 450–490 nm/ λ_em = 515–565 nm) was used and pictures of cells were taken with an exposure time of 5 s.