Recombinant expression and purification of wheat aminoacyl-tRNA synthetases and IRES-dependent polypeptide synthesis with homogeneously derived purified factors

Abstract

Reconstitution of the translation system including the full set of aminoacyl-tRNA synthetases (ARSs) has been achieved with the components from Escherichia coli and human. We are trying to reconstitute the plant translation system because plants are also essential targets of biotechnology. Some eukaryotic ARSs form multi-synthetase complexes (MSCs), while plant MSCs have not been fully characterized by the conventional top-down approaches isolating them from plant tissues. To reveal more about the plant MSCs by bottom-up approaches and to reconstitute the plant translation system, we attempted here to prepare individual wheat ARSs by E. coli expression methods and sequenced tRNAs expressed in wheat germs. The 16 ARSs other than CysRS, IleRS, LysRS and ThrRS were synthesized in E. coli and were purified to near homogeneity. Fourteen of them other than ValRS and AsnRS had their aminoacylation activity. Then, the 6 ARSs that were not prepared successfully in E. coli (ValRS, AsnRS, IleRS, ThrRS, LysRS, and CysRS) were synthesized in a wheat-germ cell-free translation system and purified. As a result, ThrRS and ValRS, but not the other four, could aminoacylate wheat germ tRNA. Some of the unsuccessful ARSs might require unidentified co-translational interactions for formation of their active forms. Finally, a reconstituted wheat translation system containing 8 ARSs, eEFs, eRFs, ribosome, and total tRNA was constructed. The system successfully translated an mRNA encoding a hemagglutinin-tag and a randomly generated sequence of 5 amino acids downstream of a cricket paralysis virus internal ribosome entry site, which enables translation independent of initiation factors.