Residue-specific incorporation of phenylalanine analogues into protein biosynthesis in silkworm cultured cells

Abstract

Here we describe the residue-specific incorporation of para-substituted L-phenylalanine (Phe) analogues into protein biosynthesis in Bombyx mori cultured cells, BmN, which express B. mori phenylalanyl-tRNA synthetase (BmPheRS) mutants with relaxed amino acid specificity. Aminoacylation capabilities of BmPheRS mutants (Ala450 to Gly and Thr407 to Ala or Gly mutants in the α-subunit) were first investigated in vitro and then the incorporation of Phe analogues into a reporter protein, EGFP, was investigated using BmN cells expressing one of the above mutants. We also evaluated the effects of lowering the Phe concentration in culture media, which competes with Phe analogues in aminoacylation reactions inside cells, and found that lowering Phe concentration was effective in increasing the substitution rate from Phe to its analogues in EGFP. We conducted electrophoretic and mass spectrometric analyses of synthesized EGFP to clarify the incorporation of Phe analogues. We obtained direct mass spectrometric data demonstrating the incorporation of p-bromo- and p-cyano-L-phenylalanine and indirect electrophoretic data, implying the incorporation of p-azido- and p-iodo-L-phenylalanine into protein biosynthesis in BmN cells.