海南捕鸟蛛毒素-Ⅳ在大肠杆菌中的分泌表达及纯化  

Extracellular Expression and Purification of Spider Neurotoxin Hainantoxin-Ⅳ in Escherichia coli

作　　者：刘长军 王健杰 易可 张哲扬 李慧敏 严青 孟尔 LIU Chang-Jun;WANG Jian-Jie;YI Ke;ZHANG Zhe-Yang;LI Hui-Min;YAN Qing;MENG Er(Department of Bioengineering,School of Life and Health Sciences,Hunan University of Science and Technology,Xiangtan 411201,Hunan,China;Key Laboratory of Genetic Improvement and Multiple Utilization of Economic Crops in Hunan Province,Hunan University of Science and Technology,Xiangtan 411201,Hunan,China;Key Laboratory of Ecological Remediation and Safe Utilization of Heavy Metal-polluted Soils,Hunan University of Science and Technology,Xiangtan 411201,Hunan,China)

机构地区：[1]湖南科技大学生命科学与健康学院生物工程系,湖南湘潭411201 [2]湖南科技大学经济作物遗传改良与综合利用湖南省重点实验室,湖南湘潭411201 [3]湖南科技大学重金属污染生态土壤修复与安全利用湖南省高校重点实验室,湖南湘潭411201

出　　处：《中国生物化学与分子生物学报》2023年第8期1191-1199,共9页Chinese Journal of Biochemistry and Molecular Biology

基　　金：湖南省教育厅基金优秀青年项目(No.22B0482);湖南科技大学博士启动基金(No.E51992,No.E51993);国家大学生创新创业训练计划项目(No.S202110534033)资助。

摘　　要：海南捕鸟蛛毒素-Ⅳ(hainantoxin-Ⅳ,HNTX-Ⅳ)是从海南捕鸟蛛(Selenocosmia hainana)的毒液中分离得到的一种可以特异性抑制河豚毒素敏感型(TTX-S)钠通道的多肽毒素,并且能够选择性抑制Nav1.7亚型电压门控钠离子通道。HNTX-Ⅳ的成熟肽含有35个氨基酸残基,其结构通过3对二硫键稳定。HNTX-Ⅳ是研究钠通道结构和功能以及镇痛药物开发过程中非常有用的一种分子探针,但是其在天然毒素中的含量较少,很难分离足够量的组分进行后续研究。在本研究中,我们通过OEPR克隆构建了HNTX-Ⅳ的大肠杆菌分泌表达质粒pSE-G1M5-SUMO-HNTX-Ⅳ。将质粒pSE-G1M5-SUMO-HNTX-Ⅳ转化到大肠杆菌BL21(DE3)中进行自动诱导分泌表达,含有6×His标签和SUMO标签的融合蛋白质6×His-SUMO-HNTX-Ⅳ成功分泌到大肠杆菌的细胞外培养基中。通过弱阳离子交换柱富集和Ni-NTA柱纯化之后,成功获得了纯度较高的融合蛋白质6×His-SUMO-HNTX-Ⅳ。通过Ulp1激酶切割,重组HNTX-Ⅳ(rHNTX-Ⅳ)被释放出来,并通过RP-HPLC分离获得。通过MALDI-TOF质谱鉴定rHNTX-Ⅳ分子量为3.9876kD。通过膜片钳技术,10μmol/L的rHNTX-Ⅳ能够抑制90%以上的hNav1.7的电流,其IC_(50)值为126 nmol/L。本研究成功将HNTX-Ⅳ分泌到了大肠杆菌的细胞外培养液中,并最终获得了纯度较高且具有生理活性的rHNTX-Ⅳ分子,丰富了HNTX-Ⅳ的制备方法,为其后续研究奠定了基础。The spider neurotoxin hainantoxin-Ⅳ(HNTX-Ⅳ),which is isolated from the crude venom of the spider Selenocosia hainana,can specifically inhibit the tetrodotoxin-sensitive(TTX-S)sodium channel,and can selectively inhibit Voltage-gated sodium channel(VGSC)Nav1.7.The mature peptide of HNTX-Ⅳ contains 35 amino acid residues connected by three pairs of disulfide bonds.HNTX-Ⅳ is con-sidered as a useful molecular probe for investigating the structure and function of VGSCs and developing analgesics.Due to its low abundance in crude venom,it is difficult to separate sufficient HNTX-Ⅳ for subsequent research and development experiments.In this study,a secretory expression plasmid pSE-G1M5-SUMO-HNTX-Ⅳ for extracellular expression of HNTX-Ⅳ in Escherichia coli was constructed by u-sing OEPR cloning and transformed into BL21(DE3).After auto-induction expression,the fusion protein 6×His-SUMO-HNTX-Ⅳ containing a 6×His tag and a SUMO tag was successfully secreted into the extra-cellular medium.After enrichment with weak-cation-exchange chromatography and purification with Ni-NTA column,the fusion protein 6×His-SUMO-HNTX-Ⅳ with high purity was successfully obtained.Af-ter digestion with Ulp1 kinase,recombinant HNTX-Ⅳ(rHNTX-Ⅳ)was released and subsequently sepa-rated by RP-HPLC.The molecular weight of rHNTX-Ⅳ was identified as 3.9876kD by MALDI-TOF mass spectrometry.With patch clamp technology,10μmol/L of rHNTX-Ⅳ could inhibit more than 90%of hNav1.7,and its IC_(50)value was 126 nmol/L.In conclusion,rHNTX-Ⅳ was successfully secreted into extracellular space of E.coli for the first time,and an alternative method for the preparation of HNTX-Ⅳ was confirmed.

关 键 词：海南捕鸟蛛毒素-Ⅳ 细胞外分泌表达 大肠杆菌 

分 类 号：Q78[生物学—分子生物学]