Figure 2. Efficiency of amber suppression with different unnatural amino acids.

Figure 2

Efficiency of amber suppression with different unnatural amino acids. tRNAPheCUA from yeast was chemically acylated with naphthylalanine (I) and biocytin (biotinylated lysine) (II), respectively. The resulting aminoacylated amber suppressor tRNAs were used in cell-free protein biosynthesis reactions with the EasyXpress standard batch system (distributed by QIAGEN GmbH, Hilden, Germany). The gene for the Fatty Acid Binding Protein from bovine liver (FABP) carrying an amber codon at position 88 served as the template. Protein synthesis was performed in the presence of 14C leucine. Following protein synthesis and SDS-PAGE radiolabeled proteins were visualized with a Typhoon 8600 variable mode imager (GE Healthcare Europe GmbH, Munich, Germany). The efficiency of suppression strongly depends on the nature of the unnatural amino acid. With biotinylated lysine release factor 1 (RF1) mediated termination is favored, whereas suppression is predominant with naphthylalanine. w/o = control reaction without amber suppressor tRNA.

From: Cell-Free Synthesis of Defined Protein Conjugates by Site-directed Cotranslational Labeling

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