Moura-da-Silva A M, Línica A, Della-Casa M S, Kamiguti A S, Ho P L, Crampton J M, Theakston R D
Laboratório de Imunopatologia, Laboratório de Biotecnologia Molecular, Instituto Butantan, Av. Vital Brasil, 1500, São Paulo, 05503-900, Brazil.
Arch Biochem Biophys. 1999 Sep 15;369(2):295-301. doi: 10.1006/abbi.1999.1372.
Jararhagin, a hemorrhagin from Bothrops jararaca venom, is a soluble snake venom component comprising metalloproteinase and disintegrin cysteine-rich domains and, therefore, is structurally closely related to the membrane-bound A Disintegrin And Metalloproteinase (ADAMs) protein family. Its hemorrhagic activity is associated with the effects of both metalloproteinase and disintegrin domains; the metalloproteinase enzymatically damages the endothelium and the disintegrin domain inhibits platelet-collagen interactions. The expression of whole jararhagin or its disintegrin domain has never been attempted before. The aim of this study was to investigate whether we could express the disintegrin domain of jararhagin and to verify whether this domain displays an inhibitory effect on the platelet-collagen interaction. Therefore, the cDNA fragment coding for the disintegrin plus cysteine-rich domains of jararhagin was cloned into the pET32a vector, used to transform the Escherichia coli AD494(DE3)pLysS strain. The thioredoxin-disintegrin fusion protein was recovered from the soluble extract of the cells, yielding up to 50 mg/liter culture. The fusion protein was isolated using polyhistidine binding resin which resulted in a main band of 45 kDa recognized by anti-native jararhagin antibodies. Antibodies raised in rabbits against the fusion protein had high enzyme-linked immunosorbent assay titers against native jararhagin and detected a band of 52 kDa on Western blots of whole B. jararaca venom demonstrating that these antibodies recognize the parent jararhagin molecule. Treatment of the fusion protein with enterokinase, followed by further capture of the enzyme, resulted in a band of 30 kDa, the expected size for jararhagin-C. Further purification of the cleaved disintegrin using FPLC Mono-Q columns resulted in one fraction capable of efficiently inhibiting collagen-induced platelet aggregation in a dose-dependent manner (IC(50) of 8.5 microg/ml).
矛头蝮蛇毒出血毒素（jararhagin）是一种从巴西矛头蝮蛇毒中提取的出血毒素，是一种可溶性蛇毒成分，由金属蛋白酶和富含半胱氨酸的解整合素结构域组成，因此在结构上与膜结合的解整合素金属蛋白酶（ADAMs）蛋白家族密切相关。其出血活性与金属蛋白酶和解整合素结构域的作用有关；金属蛋白酶通过酶促作用损伤内皮细胞，解整合素结构域抑制血小板与胶原蛋白的相互作用。此前从未尝试过表达完整的矛头蝮蛇毒出血毒素或其解整合素结构域。本研究的目的是探究我们是否能够表达矛头蝮蛇毒出血毒素的解整合素结构域，并验证该结构域是否对血小板与胶原蛋白的相互作用具有抑制作用。因此，将编码矛头蝮蛇毒出血毒素解整合素加富含半胱氨酸结构域的cDNA片段克隆到pET32a载体中，用于转化大肠杆菌AD494(DE3)pLysS菌株。硫氧还蛋白-解整合素融合蛋白从细胞的可溶性提取物中回收，每升培养物产量高达50毫克。使用多组氨酸结合树脂分离融合蛋白，得到一条45 kDa的主带，可被抗天然矛头蝮蛇毒出血毒素抗体识别。用兔制备的针对融合蛋白的抗体对天然矛头蝮蛇毒出血毒素具有高酶联免疫吸附测定效价，并在整个巴西矛头蝮蛇毒的蛋白质印迹上检测到一条52 kDa的条带，表明这些抗体识别亲本矛头蝮蛇毒出血毒素分子。用肠激酶处理融合蛋白，随后进一步捕获该酶，得到一条30 kDa的条带，这是矛头蝮蛇毒出血毒素-C的预期大小。使用快速蛋白质液相色谱法（FPLC）Mono-Q柱进一步纯化切割后的解整合素，得到一个能够以剂量依赖性方式有效抑制胶原蛋白诱导的血小板聚集的组分（半数抑制浓度（IC50）为8.5微克/毫升）。
