Expression, purification and crystallization of a lyssavirus matrix (M) protein

Abstract

The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 A resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6(1)22 or P6(5)22, with unit-cell parameters a = b = 56.9-57.2, c = 187.9-188.6 A, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress.

Figure 1

SDS–PAGE of purified LAG M (molecular weights are indicated in kDa).

Figure 2

Crystals obtained for native and SeMet-substituted LAG M with or without ZnCl₂ added to the protein. The scale bar corresponds to 50 µm. (a) Native LAG M, no ZnCl₂ added, crystallization conditions 10%(v/v) 2-methyl-2,4-pentanediol, 0.1 M sodium acetate pH 5.0. (b) Native LAG M, ZnCl₂ added, crystallization conditions 10%(w/v) PEG 6000, 0.1 M citrate pH 4.0. (c) SeMet-substituted LAG M, ZnCl₂ added, crystallization conditions 0.75%(w/v) PEG 6000, 0.1 M citrate pH 4.0. (d) Native LAG M, ZnCl₂ added, crystallization conditions 0.1 M citrate pH 4.0, 1.0 M sodium chloride.

Figure 3

Re-annealing of cryocooled crystals greatly enhanced diffraction quality. Identical diffraction images (Δϕ = 0.5°) were collected from a single crystal of SeMet LAG M (a) before and (b) after re-annealing the crystal by removing it from the cold nitrogen-gas cryostream, quickly sweeping it through the cryoprotectant solution and then re-cryocooling in the cryostream.