A Nature research paper describes use of synthetic peptides to disrupt Ca2+-triggered membrane fusion. The finding could guide new therapies for diseases caused by mucus hypersecretion
strain BL-21 with an N-terminal, TEV-protease-cleavable hexa-histidine tag. The expression and purification protocols were mostly identical to that of Stx1A. The protein was expressed overnight at 30 °C in 8 l of autoinducing medium. Cell pellets from 8 l of culture were suspended in 1× PBS, 1 mM EDTA, 1 mM PMSF and 8 EDTA-free protease inhibitor tablets supplemented with lysozyme and DNase I . The cells were lysed using a sonicator and an M-110EH microfluidizer .
5 h. The solubilized membrane was centrifuged at 43,000 rpm for 35 min, the supernatant was mixed with 5 ml of Glutathione Sepharose 4B and incubated overnight at 4 °C with end-over-end mixing. The beads were washed with 20 CV of 20 mM HEPES pH 7.5, 300 mM NaCl, 2 mM DTT, 1 mM EDTA and 110 mM OG. The protein was cleaved off the column by resuspending the beads in 2 ml of wash buffer supplemented with 110 µg of TEV protease, and incubating at 4 °C for 1 h.
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