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Recently, we have succeeded in incorporating various nonnatural amino acids into proteins by using four-base codon-anticodon pairs in Spodoptera frugiperda 21 (Sf21) insect cell-free protein synthesizing system. Here, the reaction was conducted under various conditions in order to optimize the incorporation efficiency. The optimal concentration of aminoacyl-tRNA, reaction temperature, and reaction time were 2 nM, 25 degrees C, and 1.5 hr, respectively.

Efficient incorporation of a nonnatural amino acid into a protein in an insect cell-free translation system