High-level production and isotope labeling of snake neurotoxins, disulfide-rich proteins
High-level production and isotope labeling of snake neurotoxins, disulfide-rich proteins
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DOI：
10.1006/prep.1997.0740
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发表时间：
1997-08-01
影响因子：
1.6
通讯作者：
Menez, A
中科院分区：
文献类型：
--
作者：
Drevet, P;Lemaire, C;Menez, A
这项工作的目的是生产和标记蛇神经毒素，富含二硫化物的蛋白质。蛇毒素的突变体erabutoxin a被用作模型。其N-末端部分与蛋白A（B）的合成IgG结合结构域ZZ融合。Nilsson et QI.，1987，Protein Eng.1，107-113），从而防止在细菌细胞质中的降解，并提供了一种在IgG琼脂糖上的简单亲和纯化方法。获得了可溶性融合蛋白，产量为60 mg/L，相当于20 mg/L毒素。毒素部分在柱上折叠，而杂合体仍然结合。对复性的氧化还原条件进行了优化，发现是氧化性的，但需要还原分子。与柱结合的杂交体的浓度可以增加至3.3mg/ml，而不显著改变折叠过程。CNBr裂解融合蛋白，然后进行纯化步骤，每克干细胞重量产生约2 mg生物活性毒素突变体。应用该方法生产55 mg均匀标记有N-15的毒素。(C)北京：科学出版社.
The aim of this work was to produce and to label snake neurotoxins, disulfide-rich proteins. A mutant of a snake toxin, erabutoxin a, was used as a model. Its N-terminal part was fused to ZZ, a synthetic IgG-binding domain of protein A (B. Nilsson et QI., 1987, Protein Eng. 1, 107-113), thus preventing degradation in the bacterial cytoplasm and providing a simple affinity-purification method on IgG Sepharose. A soluble fusion protein was obtained with a yield of 60 mg/L, corresponding to 20 mg/L toxin. The toxin moiety was folded on the column while the hybrid was still bound. The oxidoreducing conditions for the refolding were optimized and were found to be oxidative but With a need for reducing molecules. The concentration of the hybrid bound to the column could be increased up to 3.3 mg/ml without significantly altering the folding process. CNBr cleavage of the fusion protein followed by a purification step yielded about 2 mg of biologically active toxin mutant per gram of dry cell weight. This procedure was applied to produce 55 mg of a toxin uniformly labeled with N-15. (C) 1997 Academic Press.