Purification and biochemical characterization of a monomeric form of papaya mosaic potexvirus coat protein

Papaya mosaic virus (PapMV) is a flexuous rod shape virus made of 1400 subunits that assemble around a plus sense genomic RNA. The structure determination of PapMV and of flexuous viruses in general is a major challenge for both NMR and X-ray crystallography. In this report, we present the characterization of a truncated version of the PapMV coat protein (CP) that is suitable for NMR study. The deletion of the N-terminal 26 amino acids of the PapMV CP (CP27–215) generates a monomer that can be expressed to high level and easily purified for production of an adequate NMR sample. The RNA gel shift assay showed that CP27–215 lost its ability to bind RNA in vitro, suggesting that the multimerization of the subunit is important for this function. The fusion of a 6× His tag at the C-terminus improved the solubility of the monomer and allowed its concentration to 0.2 mM. The CD spectra of the truncated and the wild-type proteins were similar, suggesting that both proteins are well ordered and have a similar secondary structure. CP27–215 was 15N labeled for NMR studies and a 2D 1H–15N-HSQC spectrum confirmed the presence of a well-ordered structure and the monomeric form of the protein. These results show that CP27–215 is amenable to a complete and exhaustive NMR study that should lead to the first three-dimensional structure determination of a flexuous rod shape virus.