Expression, purification, and functional characterization of an N-terminal fragment of the tomato mosaic virus resistance protein Tm-1

Tm-1, the protein product of Tm-1, a semidominant resistance gene of tomato, inhibits tomato mosaic virus (ToMV) replication by binding to ToMV replication proteins. Previous studies suggested the importance of the Tm-1 N-terminal region for its inhibitory activity; however, it has not been determined if the N-terminal region is sufficient for inhibition. Furthermore, the three-dimensional structure of Tm-1 has not been determined. In this study, an N-terminal fragment of Tm-1 (residues 1–431) as a fusion protein containing an upstream maltose-binding protein was expressed in E. coli Rosetta (DE3) cells at 30 °C and then purified. The solubility of the fusion protein was greater when the cells were cultured at 30 °C than when cultured at lower or higher temperatures. The purified N-terminal Tm-1 fragment from which the maltose-binding protein tag had been removed has inhibitory activity against ToMV RNA replication.

► A fusion protein containing the maltose-binding protein and an N-terminal region of Tm-1 was expressed in Escherichia coli. ► The solubility of the protein was greatly influenced by the induction temperature. ► The purified fragment is likely a homodimer and inhibits ToMV RNA replication.