Figure 3. IRES-mediated cell-free synthesis of EGFR-eYFP-AzF687 by amber suppression using an orthogonal tRNA/synthetase pair and subsequent chemo-selective fluorescence modification.

(a) eYFP fluorescence obtained using the standard template (−IRES) and the IRES template with an amber codon at position 687 (+IRES) in the absence (−PG) and the presence of poly G (+PG). Error bars represent the standard deviation of triplicate analysis. (b) Autoradiography and (c) in-gel fluorescence of microsomal fractions from reactions performed using the IRES template without (WT) and with amber codon (Amb) in the presence of poly G after treatment with DyLight650 phosphine. Isotopic labeling was achieved by ¹⁴C-leucine supplementation. NTC: control reaction without DNA template.