在大肠杆菌中产生的多组氨酸标签的东亚钳蝎抗肿瘤镇痛肽的体外重折叠及功能分析
In vitro refolding and functional analysis of polyhistidine-tagged Buthus martensii Karsch antitumor-analgesic peptide produced in Escherichia coli.
作者信息
Cao Qingxin, Lu Wuguang, Cai Xueting, Hu Chunping, Wang Chen, Ye Juan, Yan Huaijiang, Yang Yang, Wang Zhigang, Huo Jiege, Liu Yan, Yu Ye, Ling Changquan, Cao Peng
机构信息
Changhai Hospital of Traditional Chinese Medicine, Second Military Medical University, Shanghai, 200433, China.
, 455 Military Hospital, Shanghai, 200052, China.
出版信息
Biotechnol Lett. 2015 Dec;37(12):2461-6. doi: 10.1007/s10529-015-1936-8. Epub 2015 Aug 25.
OBJECTIVES
To identify an efficient in vitro refolding method to generate highly active His6-tagged scorpion toxin antitumor-analgesic peptide (AGAP) isolated from Escherichia coli inclusion bodies.
RESULTS
N- and C-Terminal His6-tagged recombinant (r) AGAP (NHis6-rAGAP and CHis6-rAGAP, respectively) were expressed in E. coli; the purification and refolding conditions were optimized. CHis6-rAGAP, but not NHis6-rAGAP, exhibited significant in vitro antihepatoma activity that was much greater than that of rAGAP produced using SUMO fusion technology (IC50, 0.4 ± 0.08 vs. 1.8 ± 0.3 μM). CHis6-rAGAP also showed significant inhibition of tumor growth in a mouse xenograft model of human hepatoma and inhibition of neuronal excitability, demonstrated by blockage of voltage-sensitive tetrodotoxin-resistant (TTX-R) sodium currents in acute isolated dorsal root ganglion neurons.
CONCLUSIONS
This refolding protocol optimized for C-terminal His6-tagged scorpion rAGAP is potentially applicable to similar long-chain and cysteine-rich toxins.
目的
鉴定一种高效的体外重折叠方法，以产生从大肠杆菌包涵体中分离得到的具有高活性的His6标签蝎毒素抗肿瘤镇痛肽（AGAP）。
结果
N端和C端带有His6标签的重组（r）AGAP（分别为NHis6-rAGAP和CHis6-rAGAP）在大肠杆菌中表达；优化了纯化和重折叠条件。CHis6-rAGAP而非NHis6-rAGAP表现出显著的体外抗肝癌活性，其活性远高于使用SUMO融合技术产生的rAGAP（IC50，0.4±0.08对1.8±0.3μM）。CHis6-rAGAP在人肝癌小鼠异种移植模型中也显示出对肿瘤生长的显著抑制作用，并抑制神经元兴奋性，这通过急性分离背根神经节神经元中电压敏感性抗河豚毒素（TTX-R）钠电流的阻断得以证明。
结论
这种针对C端His6标签蝎毒素rAGAP优化的重折叠方案可能适用于类似的长链和富含半胱氨酸的毒素。
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