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Recently, we succeeded in incorporating a biotin tag into an active protein, but only at the N-terminal site, in the presence of an Escherichia coli initiator tRNA(fMet) aminoacylated with methionine biotinylated at the alpha-amino group. The biotinylated protein was immobilized on a streptavidin-matrix. We have tried to increase the biotin labeling efficiency (immobilization efficiency) by decreasing concentrations of non-biotinylated tRNA(fMet)s in the translation system.

A novel immobilization method of an active protein via in vitro N-terminal specific incorporation system of nonnatural amino acids