Cloning of Bombyx mori phenylalanyl-tRNA synthetase and the generation of its mutant with relaxed amino acid specificity

抄録

The genes encoding two subunits (α and β) of phenylalanyl-tRNA synthetase (PheRS), which catalyzes aminoacylation of tRNAPhe with L-phenylalanine (Phe), were cloned from the silk glands of the domesticated silkworm, Bombyx mori, and their full-length cDNA sequences were identified. The deduced amino acid sequences of the B. mori PheRS (BmPheRS) showed a high similarity with its counterparts from other organisms. A comparison with the previous mutation studies suggested that the Ala residue at the amino acid position 450 in the α-subunit of BmPheRS would be one of the key determinants for discriminating Phe from its amino acid competitors. To relax the amino acid specificity of BmPheRS, Ala to Gly mutation was introduced at the residue 450 of its α-subunit to generate the αA450G BmPheRS mutant. In vitro activity assay demonstrated that the recombinant αA450G BmPheRS mutant catalyzed the aminoacylation of the synthesized B. mori tRNAPhe with p-chloro- and p-bromo-substituted Phe analogs which were not recognized by the wild-type BmPheRS. This finding opens up the possibility of developing a novel type of genetically-modified silkworm which can utilize unnatural amino acids as protein building blocks.