The complex between SOS3 and SOS2 regulatory domain from Arabidopsis thaliana: cloning, expression, purification, crystallization and preliminary X-ray analysis

Abstract

The salt-tolerance genes SOS3 (salt overly sensitive 3) and SOS2 (salt overly sensitive 2) regulatory domain of Arabidopsis thaliana were cloned into a polycistronic plasmid and the protein complex was expressed in Escherichia coli, allowing purification to homogeneity in three chromatographic steps. Crystals were grown using vapour-diffusion techniques. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 44.14, b = 57.39, c = 141.90 A.

Figure 1

Expression and purification of the SOS3Δ15–SOS2T1 complex. Lane 1, flowthrough from the anion-exchange column. Lane 2, flowthrough from the cation-exchange column. Lane 3, the fraction eluted with 125 mM NaCl buffer from the cation-exchange column. Lane 4, the main peak from the permeability column. Proteins were analyzed using 15% SDS–PAGE gels. Gels were stained using Coomassie Brilliant Blue.

Figure 2

Gel-filtration chromatography of SOS3Δ15–SOS2T1. The line shows the absorbance recorded at 280 nm.

Figure 3

(a) SOS3Δ15–SOS2T1 crystals grown in 20%(w/v) PEG 4000, 0.1 M Tris–HCl pH 8.5, 0.2 M lithium sulfate. (b) Effect of ethylene glycol and guanidine–HCl on the crystals used in the study.