Bombyx mori silk fibroin incorporating three methionine (Met) analogues-homopropargylglycine (Hpg), azidohomoalanine (Aha), and homoallylglycine (Hag)-can be produced simply by adding them to the diet of B. mori larvae. The Met analogues are recognized by methionyl-tRNA synthetase, bound to tRNA(Met), and used for the translation of adenine-uracil-guanine (AUG) codons competitively with Met. In the presence of the standard amount of Met in the diet, incorporation of these analogues remains low. Lowering the amount of Met in the diet drastically improves incorporation efficiencies. Alkyne and azide groups in Hpg and Aha incorporated into silk fibroin can be selectively modified with Cu-catalyzed azide-alkyne cycloaddition reactions (click chemistry). Since Met residues exist only at the N-terminal domain of the fibroin heavy chain and in the fibroin light chain, good access to the reactive sites is expected and domain-selective modifications are possible without perturbing other major domains, including repetitive domains.
Keywords: click chemistry; modification; proteins; silk fibroin; unnatural amino acids.
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