Extended Data Fig. 2: Heterologously produced VdAve1 can be isolated from inclusion bodies.
From: Microbiome manipulation by a soil-borne fungal plant pathogen using effector proteins
a, E. coli BL21 cells were grown in liquid YT medium and VdAve1 expression was induced using 1 mM IPTG. Following four hours of protein production at 28oC, the presence of VdAve1 was confirmed by boiling the cells in 1% SDS, 2M urea, 1.25% β-mercaptoethanol, 2.5% glycerol, 15 mM Tris, pH 6.8. The band representing VdAve1 is indicated with an asterisk; limited solubility of the protein was detected upon sonication of the cells in the corresponding buffers (lanes 1-9). The presence of VdAve1 in the insoluble protein fractions was confirmed following denaturation of the insoluble proteins, indicating the formation of inclusion bodies. The experiment has been repeated three times with similar results, the gel image is from a single experiment. b, VdAve1 purified from the insoluble protein fraction under denaturing conditions was refolded by step-wise dialysis. Functionality of the protein was confirmed by infiltration into N. tabacum leaf sections overexpressing the corresponding tomato immune receptor Ve1, resulting in a hypersensitive response at three days post infiltration.
