High Yield Production and Refolding of the Double-Knot Toxin, an Activator of TRPV1 Channels
(A) Purification of linear DkTx. Cleavage of the fusion protein and reduction of the disulfide bond were accomplished using hydroxylamine and DTT, respectively, as described in methods. (B) Linear DkTx was folded in 1 M NH4OAc (pH 8.0) buffer containing 1 M GdnHCl, 1 mM EDTA, 2.5 mM GSH and 0.25 mM GSSG for 5 days at 4°C. The toxins were purified using a linear gradient of 29–44% solvent B for 15 min at a flow rate of 14 ml/min, where solvent A was water containing 0.1% TFA and solvent B was acetonitrile containing 0.1% TFA. (C) Fraction 5 activated TRPV1 expressed in oocytes. Holding voltage was −60 mV.