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. 2022 Oct 4;13:5856. doi: 10.1038/s41467-022-33516-1

Fig. 7. Purification and antimicrobial activity of SUMO-AMP fusion proteins from transplastomic plants.

Fig. 7

a Ni-NTA agarose purification of SUMO-AMP and SUMO-GFP fusion proteins based on the 6xHis tag. Protein samples were separated by gel electrophoresis and blotted. The western blot was stained with Coomassie prior to blocking and immunodetection with an anti-HA antibody. For extraction, frozen leaf material was ground and mixed with extraction buffer. An aliquot of this suspension was directly loaded as total protein (To; incubated with denaturing sample buffer for 5 min at 95 °C and insoluble material removed by centrifugation). The insoluble material was then removed by centrifugation, and the supernatant was used as input for purification (In) by incubation with Ni-NTA agarose beads. The unbound flow through fraction was collected (Ft), and after washing the column, the bound SUMO fusion proteins were eluted (E). Asterisks indicate the position of the fusion proteins in the To and In fractions. Two independent blots were performed with similar results. b Cleavage assay of the purified SUMO fusion proteins using SUMO protease. 170 µg SUMO-GFP (S-GFP), 45 µg SUMO-HA-WAM1 (S-W), and 96 µg SUMO-HA-Novispirin (S-N) were incubated with (+) or without (−) 25 units of SUMO protease (Ulp1: 27 kDa). Of these samples, 35.5 µg SUMO-GFP, 9 µg SUMO-HA-WAM1, and 20 µg SUMO-HA-Novispirin were separated by SDS-PAGE followed by western blotting. The resulting membranes were stained with Coomassie followed by immunodetection of S-W and S-N with an anti-HA antibody. Two independent blots were performed with similar results. c Activity test of purified AMPs by radial diffusion assays. Equimolar concentrations (150 µM) of SUMO fusions (in 20 mM Tris pH 8.0 buffer) were incubated with or without 2.5 units of SUMO protease, and applied in an antimicrobial activity radial diffusion assay. Antimicrobial activity is observed as a clear zone without bacterial growth around the well. The well in the center of the plate was loaded with the buffer control. Three independent assays were performed with similar results. For abbreviations, see (b). Source data are provided as a Source Data file.