A secretory system for extracellular production of spider neurotoxin huwentoxin-I in Escherichia coli

被引:9
|
作者
Liu, Changjun [1 ,2 ,3 ]
Yan, Qing [1 ]
Yi, Ke [1 ]
Hu, Tianhao [1 ]
Wang, Jianjie [1 ]
Zhang, Zheyang [1 ]
Li, Huimin [1 ]
Luo, Yutao [1 ]
Zhang, Dongyi [1 ]
Meng, Er [1 ,2 ,3 ]
机构
[1] Hunan Univ Sci & Technol, Sch Life & Hlth Sci, 2 Taoyuan Rd, Xiangtan 411201, Hunan, Peoples R China
[2] Hunan Univ Sci & Technol, Key Lab Genet Improvement & Multiple Utilizat Econ, Xiangtan, Hunan, Peoples R China
[3] Hunan Univ Sci & Technol, Key Lab Ecol Remediat & Safe Utilizat Heavy Met po, Xiangtan, Hunan, Peoples R China
来源
PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY | 2023年 / 53卷 / 08期
关键词
Auto induction; cysteine-rich peptides; Escherichia coli; extracellular space; Huwentoxin-I; secretory expression; DISULFIDE BOND FORMATION; PROTEIN-PRODUCTION; VENOM PEPTIDES; EXPRESSION; AUTOINDUCTION; PURIFICATION; TOXINS;
D O I
10.1080/10826068.2022.2158473
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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 Escherichia coli 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 x 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 x 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 hNa(v)1.7 could be inhibited by rHWTX-I and the IC50 value was 419 nmol/L.
引用
收藏
页码:914 / 922
页数:9
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