RP-HPLC chromatography and mass spectra of rHWTX-I.

<p>A) RP-HPLC chromatography of rHWTX-I (upper) and native HWTX-I (lower) in the same C18 column. Elution was monitored at 280 nm. rHWTX-I and native HWTX-I were eluted at an acetonitrile concentration of 30.4% and 30.6%, respectively. B) Mass spectra of purified rHWTX-I. The calculated theoretical molecular weight of rHWTX-I was 3756.45 Da and the measured molecular weight was 3750.69 Da.</p

Characterization of HWTX-I.

<p>A) Amino acid sequence and disulfide bonds (black lines) of HWTX-I. B) 3D structure of HWTX-I (PDB entry 1qk6). All of the cysteines and disulfide bridges are shown in red. C) Model of the surface calculation of HWTX-I (version 0.99 beta37 by PyMOL).</p

Effects of rHWTX-I and HWTX-I on wild-type hNav_1.7 channels expressed in HEK293 cells.

<p>A) Effects of rHWTX-I (left) or native HWTX-I (right) on currents through hNav_1.7 channels that were elicited by depolarization to −10 mV from a holding potential of −100 mV. The toxin concentration was 1 µmole/L. B) Effects of rHWTX-I (left) or native HWTX-I (right) on dose-response inhibitory curves of hNav_1.7 channels. Data points shown as mean ± S.E. (each from three to five experimental cells). The IC₅₀ values of hNav_1.7 in response to rHWTX-I and native HWTX-I were 640 nmole/L and 630 nmole/L, respectively.</p

SDS-PAGE analysis.

<p>A) Lane M, protein molecular weight markers; lane 1, uninduced cell sample; line 2, induced cell sample; line3, total soluble proteins extracted from the periplasm of induced cells; line 4, purified fusion protein (arrow indicates DsbC-rHWTX-I). B) Lane M, protein molecular weight markers; lane 5, 20 µl DsbC-rHWTX-I after enterokinase digestion; lane 6, 100 µl DsbC-rHWTX-I after enterokinase digestion; line 7, negative control (10 µl DsbC-rHWTX-I, enterokinase free).</p

Construction of the pET40b-rHWTX-I expression vector.

<p>A) pET40b-FrHWTX-I. B) pET40b-rHWTX-I.</p

Construction of pET-GS-HNTX-IV expression vector.

<p>A) Schematic diagram of RF-cloning strategy. F1: forward primer. R1: reverse primer. The portion complementary with the target gene is marked gray, and the portion complementary to the recipient vector is marked black. B) Map of pET-GS-HNTX-IV expression vector.</p

Affinity chromatography independent purification of rHNTX-IV by TCA.

<p>A) M: Protein molecular weight marker; 1–10: fractions extracted by TCA from 1% to 10%; P: precipitates. B) Relative purity (compared with lane 10) and relative yield (compared with lane 1) of rHNTX-IV under different TCA concentrations. The value was calculated by grayscale by Quantity One software. The data points (means ± S.E.) come from three independent experiments. C) Purification by RP-HPLC.</p

Identification of rHNTX-IV.

<p>A) Comparison of rHNTX-IV and native HNTX-IV on RP-HPLC. B) MALDI-TOF analysis of rHNTX-IV.</p

The kinetics curves of rHNTX-IV.

<p>A) The dose-effects curve of rHNTX-IV. The data points (means ± S.E.) are fit to the Hill equation. B) The steady-state inactivation and activation curves for TTX-S <i>I</i>_Na with 100 nM rHNTX-IV. The data points (means ± S.E.) come from at least five cells and are fit to the Boltzmann equation.</p

Expression of GST-SUMO tagged HNTX-IV in BL21(DE3).

<p>A) Solubility analysis at 37°C and 16°C. M: protein molecular weight marker; S: soluble fraction. P: precipitates. The target protein is denoted by an arrow. B) The expression level under different IPTG conditions.</p