High yield production and refolding of the double-knot toxin, an activator of TRPV1 channels

Abstract

A unique peptide toxin, named double-knot toxin (DkTx), was recently purified from the venom of the tarantula Ornithoctonus huwena and was found to stably activate TRPV1 channels by targeting the outer pore domain. DkTx has been shown to consist of two inhibitory cysteine-knot (ICK) motifs, referred to as K1 and K2, each containing six cysteine residues. Beyond this initial characterization, however, the structural and functional details about DkTx remains elusive in large part due to the lack of a high yielding methodology for the synthesis and folding of this cysteine-rich peptide. Here, we overcome this obstacle by generating pure DkTx in quantities sufficient for structural and functional analyses. Our methodology entails expression of DkTx in E. coli followed by oxidative folding of the isolated linear peptide. Upon screening of various oxidative conditions for optimizing the folding yield of the toxin, we observed that detergents were required for efficient folding of the linear peptide. Our synthetic DkTx co-eluted with the native toxin on HPLC, and irreversibly activated TRPV1 in a manner identical to native DkTx. Interestingly, we find that DkTx has two interconvertible conformations present in a 1∶6 ratio at equilibrium. Kinetic analysis of DkTx folding suggests that the K1 and K2 domains influence each other during the folding process. Moreover, the CD spectra of the toxins shows that the secondary structures of K1 and K2 remains intact even after separating the two knots. These findings provide a starting point for detailed studies on the structural and functional characterization of DkTx and utilization of this toxin as a tool to explore the elusive mechanisms underlying the polymodal gating of TRPV1.

Figure 1. DNA construct encoding DkTx.

(A) Design of a synthetic DkTx gene. (B) Sequence of the toxins used in the experiments. Note that DkTx has an N-terminal Gly residue because of the hydroxyl amine cleavage of Asn-Gly sequence added to the N-terminus.

Figure 2. Expression of DkTx in E. coli.

SDS-PAGE gels (15%) were stained with Coomassie Blue. Lane M, molecular weight markers (kDa); lane T, total cell lysate; lane P, pellet fraction; lane S, soluble fraction.

Figure 3. Purification and activity of the DkTx folding product.

(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.

Figure 4. HPLC profiles of DkTx during the folding reaction.

The peptides were separated using 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. (A) Oxidative folding of DkTx in redox buffer was monitored by HPLC. Asterisks indicate correctly folded DkTx. (B) HPLC chromatogram of purified DkTx. Dashed and solid arrows indicate the minor and major forms of DkTx, respectively. (C) The minor form was collected and re-injected after 75 min. (D) The major form was collected and re-injected after 175 min. (E) Co-injection of native and synthetic DkTx.

Figure 5. Folding kinetics of K1, K2 and DkTx.

(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.

Figure 6. Activity of K1, K2 and DkTx on TRPV1 channels.

(A) Synthetic K1, K2 and DkTx activate TRPV1 channels. Oocytes were held at −60 mV and toxins were added to the recording chamber. (B) Concentration-dependence for activation of TRPV1 by the toxins.

Figure 7. CD spectra of K1, K2 and DkTx.

The CD spectra were recorded in 0.01 M sodium phosphate (pH 7.0) at 20°C. K1+K2 depicts the added values of the CD spectra from K1 and K2.