School of Life and Health Sciences, Hunan University of Science and Technology, Xiangtan, Hunan, PR China.
Key Laboratory of Genetic Improvement and Multiple Utilization of Economic Crops in Hunan Province, Hunan University of Science and Technology, Xiangtan, Hunan, PR China.
Prep Biochem Biotechnol. 2023;53(8):914-922. doi: 10.1080/10826068.2022.2158473. Epub 2022 Dec 26.
Due to their advantages in structural stability and versatility, cysteine-rich peptides, which are secreted from the venom glands of venomous animals, constitute a naturally occurring pharmaceutical arsenal. However, the correct folding of disulfide bonds is a challenging task in the prokaryotic expression system like due to the reducing environment. Here, a secretory expression plasmid pSE-G1M5-SUMO-HWTX-I for the spider neurotoxin huwentoxin-I (HWTX-I) with three disulfides as a model of cysteine-rich peptides was constructed. By utilizing the signal peptide G1M5, the fusion protein 6 × His-SUMO-HWTX-I was successfully secreted into extracellular medium of BL21(DE3). After enrichment using cation-exchange chromatography and purification utilizing the Ni-NTA column, 6 × His-SUMO-HWTX-I was digested via Ulp1 kinase to release recombinant HWTX-I (rHWTX-I), which was further purified utilizing RP-HPLC. Finally, both impurities with low and high molecular weights were completely removed. The molecular mass of rHWTX-I was identified as being 3750.8 Da, which was identical to natural HWTX-I with three disulfide bridges. Furthermore, by utilizing whole-cell patch clamp, the sodium currents of hNa1.7 could be inhibited by rHWTX-I and the IC value was 419 nmol/L.
由于其结构稳定性和多功能性的优势，富含半胱氨酸的肽，这些肽从毒液腺的毒液动物分泌，构成了一个自然发生的药物库。然而，在原核表达系统中，由于还原环境，正确折叠二硫键是一项具有挑战性的任务。在这里，构建了一个带有三个二硫键的富含半胱氨酸肽模型蜘蛛神经毒素 huwentoxin-I（HWTX-I）的分泌表达质粒 pSE-G1M5-SUMO-HWTX-I。通过利用信号肽 G1M5，融合蛋白 6×His-SUMO-HWTX-I 成功地分泌到 BL21(DE3)的细胞外培养基中。在用阳离子交换层析进行富集和用 Ni-NTA 柱进行纯化后，6×His-SUMO-HWTX-I 通过 Ulp1 激酶消化以释放重组 HWTX-I（rHWTX-I），然后再利用反相高效液相色谱法进一步纯化。最后，完全去除了低和高分子量的杂质。rHWTX-I 的分子量鉴定为 3750.8 Da，与具有三个二硫键的天然 HWTX-I 相同。此外，通过全细胞膜片钳技术，rHWTX-I 可抑制 hNa1.7 的钠电流，IC 值为 419 nmol/L。
