用于回收聚集型小鼠肿瘤坏死因子-α的脉冲稀释法
Pulsed Dilution Method for the Recovery of Aggregated Mouse TNF-α.
作者信息
Mahmoodi Merat, Ghodsi Maryam, Moghadam Malihe, Sankian Mojtaba
机构信息
Immuno-Biochemistry lab, Immunology Research Center, Mashhad University of medical Sciences, Mashhad, Iran.
Immuno-Biochemistry lab, Immunology Research Center, Buali Research Institute , School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
出版信息
Rep Biochem Mol Biol. 2017 Apr;5(2):103-107.
BACKGROUND
The expression of mouse tumor necrosis factor alpha (TNF-α) in is a favorable way to get high yield of protein; however, the formation of cytoplasmic inclusion bodies, which is the consequence of insoluble accumulated proteins, is a major obstacle in this system. To overcome this obstacle, we used a pulsed dilution method to convert the product to its native conformation.
METHODS
Reducing agent and guanidine hydrochloride were used to solubilize inclusion bodies formed after TNF-(α) expression. Then, the refolding procedure was performed by pulsed dilution of the denatured protein into a refolding buffer. The properly-folded protein was purified by metal affinity chromatography.
RESULTS
SDS-PAGE showed a 19.9 kDa band related to the mature TNF-(α) protein. The protein was recognized by anti-mouse TNF-(α) on western blots. The final concentration of the purified recombinant TNF-(α) was 62.5 µg/mL.
CONCLUSIONS
Our study demonstrates the efficiency of this method to produce a high yield of folded mature TNF- (α).
背景
在[具体表达系统未提及]中表达小鼠肿瘤坏死因子α（TNF-α）是获得高产量蛋白质的一种有效方法；然而，不溶性积累蛋白质导致的细胞质包涵体形成是该系统中的一个主要障碍。为克服这一障碍，我们采用脉冲稀释法将产物转化为其天然构象。
方法
使用还原剂和盐酸胍溶解TNF-(α)表达后形成的包涵体。然后，通过将变性蛋白脉冲稀释到复性缓冲液中进行复性过程。正确折叠的蛋白质通过金属亲和色谱法纯化。
结果
SDS-PAGE显示出一条与成熟TNF-(α)蛋白相关的19.9 kDa条带。该蛋白在western印迹上被抗小鼠TNF-(α)识别。纯化的重组TNF-(α)的最终浓度为62.5 µg/mL。
结论
我们的研究证明了该方法在高产折叠成熟TNF-(α)方面的有效性。
Zotero 插件