Crystallographic study of Pyrococcus horikoshii tryptophanyl-tRNA synthetase

Department of Chemistry, Ochanomizu University
â—‹Akiko Mizukawa Michiko Konno

The codon for Trp is one kind and three bases of anticodon CCA of tRNATrp bind on three regions of tryptophanyl-tRNA synthetase (TrpRS), respectively. It is predicted that residues recognize bases of anticodon are conserved, since this enzyme existed from the origin of life and selects only one kind of anticodon. The comparison of the reported crystal structures between Bacillus stearothermophilus TrpRS and human TrpRS reveals difference of the orientation of an anticodon-binding α-helix domain against a catalytic domain. In order to clarify the binding region of the three bases of anticodon of tRNA through comparison with these proteins, we tried crystal structure analysis of Pyrococcus horikoshii TrpRS (Ph TrpRS).
Ph TrpRS is about 45.3kD and has 386 residues. Ph TrpRS gene was cloned into plasmid pET28c (Novagen) and over expressed in E. coli strain BL 21 (DE3) codon plus (Strategene). To remove proteins of E. coli as early as possible, supernatant of sonicated cells was heated on 70 o C for 30 minutes. Then TrpRS was purified by three steps; Ni-NTA Superflow column, RESOURCE Q column and Hi-Trap Heparin column. We tried crystallization by the hanging or sitting drop vapor diffusion technique at 20 o C and obtained about 2 μm microcrystals from drops containing 2μl of the protein (10 mg/mL), 2μl of a reservoir solution (1.6 M (NH4)2SO4, 0.2 M NaCl, 5 mM MgCl2, Tris-HCl pH 8.5-7.4) and 0.2μl of additive solution (0.1 M SrCl2).