敬钊毒素-V突变体K12A-JZTX-V的化学合成与氧化还原复性  

Solid-Phase Chemical Synthesis and Oxidative Refolding of the Mutant K12A-JZTX-V of JZTX-V

作　　者：曾雄智[1] 全妙华[2] 皮建辉[2] 梁宋平[1] 

机构地区：[1]湖南师范大学蛋白质化学与发育生物学教育部重点实验室,湖南长沙410081 [2]怀化学院生物工程系,湖南怀化418008

出　　处：《怀化学院学报》2008年第5期41-43,共3页Journal of Huaihua University

基　　金：国家自然科学基金重点项目(30430170);国家863专题(2006AA02Z141);湖南省自然科学基金项目(07JJ3072);湖南省教育厅基金项目(06C503)

摘　　要：敬钊毒素-V(Jingzhaotoxin-V,JZTX-V)是从敬钊缨毛蛛粗毒中纯化到的一种新型河豚毒素不敏感型钠通道抑制剂,由29个氨基酸残基组成,含有三对二硫键.为了深入研究该毒素的结构与功能关系,应用芴甲氧羰基(Fmoc)固相多肽化学合成方法合成了用丙氨酸(Ala)替代JZTX-V第12位赖氨酸残基的突变体K12A-JZTX-V,合成粗品经反相高效液相色谱分离纯化和MALDI-TOF质谱鉴定,证明化学合成是成功的.线性多肽经谷胱甘肽氧化复性后,复性产物经质谱测定其相对分子量为3549.63,比线性分子的理论分子质量低6,说明六个半胱氨酸已经配对形成了三对二硫键.Jingzhaotoxin- V （JZTX - V） isolated from the venom of the spider Chilobrachys jingzhao is a novel potent inhibitor that acts on tetrodotoxin - resistant sodium channels in adult rat dorsal root ganglion neurons. It is a 29 - residue polypeptide toxin including three disulfide bridges. To investigate the structure - function relationship of the toxin, a mutant of JZTX - V in which Lys 12 was substituted by Ala, was synthesized by solid- phase chemistry method with Fmoc - protected amino acids on the PS3 automated peptide synthesizer. The synthetic crude poduct was then purified by reversed - phase high performance liquid chromatography and identified by matrix- assisted laser desorption/ionization time - of- flight mass spectrometry （MALDI - TOF MASS） . The synthesis of the mutant was proved to be successful. The synthetic linear polypeptide was oxidatively refolded under the optimal conditions. The relative molecular mass of refolded K12A - JZTX - V was determined to be 3549.63 by MS. Compared with the relative molecular mass of unfolded K12A - JZTX - V （3555. 19 ）, the relative molecular mass of refolded mutant was decreased by 6 mass units, which means that all three disulfide bonds had successfully formed between six cysteine residues.

关 键 词：敬钊缨毛蛛毒素-V 固相多肽合成 突变体 氧化复性 质谱 

分 类 号：Q25[生物学—细胞生物学] Q343