Characterization of a Novel BmαTX47 Toxin Modulating Sodium Channels: The Crucial Role of Expression Vectors in Toxin Pharmacological Activity

Abstract

Long-chain scorpion toxins with four disulfide bridges exhibit various pharmacological features towards the different voltage-gated sodium channel subtypes. However, the toxin production still remains a huge challenge. Here, we reported the effects of different expression vectors on the pharmacological properties of a novel toxin BmαTX47 from the scorpion Buthus martensii Karsch. The recombinant BmαTX47 was obtained using the expression vector pET-14b and pET-28a, respectively. Pharmacological experiments showed that the recombinant BmαTX47 was a new α-scorpion toxin which could inhibit the fast inactivation of rNav1.2, mNav1.4 and hNav1.5 channels. Importantly, the different expression vectors were found to strongly affect BmαTX47 pharmacological activities while toxins were obtained by the same expression and purification procedures. When 10 µM recombinant BmαTX47 from the pET-28a vector was applied, the values of I5ms/Ipeak for rNav1.2, mNav1.4 and hNav1.5 channels were 44.12% ± 3.17%, 25.40% ± 4.89% and 65.34% ± 3.86%, respectively, which were better than those values of 11.33% ± 1.46%, 15.96% ± 1.87% and 5.24% ± 2.38% for rNav1.2, mNav1.4 and hNav1.5 channels delayed by 10 µM recombinant BmαTX47 from the pET-14b vector. The dose-response experiments further indicated the EC50 values of recombinant BmαTX47 from the pET-28a vector were 7262.9 ± 755.9 nM for rNav1.2 channel and 1005.8 ± 118.6 nM for hNav1.5 channel, respectively. Together, these findings highlighted the important role of expression vectors in scorpion toxin pharmacological properties, which would accelerate the understanding of the structure-function relationships of scorpion toxins and promote the potential application of toxins in the near future.

Voltage-Gated Sodium Channels: Structure, Function, Pharmacology, and Clinical Indications

“…Other novel scorpion toxins such as rBmαTX47, a novel α-ScTx from the scorpion Buthus martensii Karsch, have recently been cloned and expressed by Wu and Li, who also highlighted the importance of expression vectors in affecting toxin pharmacology. 115 Unfortunately, only a few structure−function studies with scorpion toxins have been undertaken because of the challenges of obtaining multimilligram quantities of these complex structures via isolation from natural sources or chemical synthesis. We envision that with the number of technological advances in the synthesis and isolation of structurally complex peptides, a new era of research in this fascinating field is just beginning.…”

“…Interestingly, because AmmVIII has a lower potency at Na v s than AaHII, Amm VIII was devoid of toxicity when injected subcutaneously to mice. Other novel scorpion toxins such as rBmαTX47, a novel α-ScTx from the scorpion Buthus martensii Karsch, have recently been cloned and expressed by Wu and Li, who also highlighted the importance of expression vectors in affecting toxin pharmacology …”

Mouse β-Defensin 3, A Defensin Inhibitor of Both Its Endogenous and Exogenous Potassium Channels

“…Then they were cracked using ultrasonic bath, the supernatant from the lysate was loaded to a nickel affinity column. The purified rmBD3 fusion protein was dialyzed with Enterokinase buffer containing 25 mM Tris-HCl, 50 mM NaCl, and 2 mM CaCl 2 for 4 h and digested by Enterokinase (Sangon Biotech, China) at the temperature of 25 °C for at least 12 h but no more than 16 h. High performance liquid chromatograph (HPLC) on a C18 column (10 × 250 mm, 5 μm) (Elite-HPLC) was used to further purify and isolate the digested protein by using a linear gradient of 5% to 95% acetonitrile with 0.1% trifluoroacetic acid (TFA) in 60 min at a constant flow rate of 4 mL/min, and the absorbance was detected at 230 nm [ 15 ]. The molecular weight was confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) [ 16 ].…”