High Yield Production and Refolding of the Double-Knot Toxin, an Activator of TRPV1 Channels
(A) HPLC chromatograms showing the folding of DkTx, K1 and K2. The left panel depicts the linear toxins. Arrows indicate correctly folded forms of the toxins. The toxins were eluted with a linear gradient of 5–65% solvent B for 30 min at a flow rate of 1 ml/min, where solvent A was water containing 0.1% TFA and solvent B was acetonitrile containing 0.1% TFA. (B) Comparison of folding kinetics of DkTx, K1 and K2. At each time point, aliquots were withdrawn and acidified by adding acetic acid. Quantification of the folding specifies was accomplished using HPLC. The correctly folded form (%) was determined based on the areas of the HPLC peaks. The curves represent fits of the data to a one-phase association.