Engineering a tRNA and aminoacyl-tRNA synthetase for the site-specific incorporation of unnatural amino acids into proteins in vivo

摘要

In an effort to expand the scope of protein mutagenesis, we have completed the first steps toward a general method to allow the site-specific incorporation of unnatural amino acids into proteins in vivo. Our approach involves the generation of an "orthogonal" suppressor tRNA that is uniquely acylated in Escherichia coli by an engineered aminoacyl-tRNA synthetase with the desired unnatural amino acid. To this end, eight mutations were introduced into tRNA_2~Gln based on an analysis of the x-ray crystal structure of the glutaminyl-tRNA aminoacyl synthetase (GlnRS)-tRNA_2~Gln complex and on pre- vious biochemical data.