A novel method for the purification of low soluble recombinant C-type lectin proteins.
A novel method for the purification of low soluble recombinant C-type lectin proteins.
复制标题
一种纯化低可溶性重组C型凝集素蛋白的新方法。
DOI：
10.1016/j.bbrc.2012.07.128
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发表时间：
2012
影响因子：
3.1
通讯作者：
Garcia,CarlosA
中科院分区：
文献类型：
--
作者：
Yin,Chunhui;Jia,Ying;Garcia,CarlosA
蛇毒含有包括蛋白质在内的许多生物分子的复杂混合物。重组蛋白的纯化是研究其功能和结构的关键步骤，亲和层析是其纯化的常用方法。在细菌表达系统中，疏水重组蛋白通常沉淀到包涵体中，纯化后的污染物通常与标记蛋白相关。本研究的目的是建立一种纯化无亲和标签的疏水重组蛋白的方法。研究了蛇毒成熟c型凝集素样蛋白（CLPs）的克隆、表达和纯化。研究了信号肽对重组蛋白表达和溶解度的影响。洗涤包涵体中的CLPs通过透析被溶解和重新折叠。无标签CLPs的纯化率是传统方法的38倍，抑制全血血小板聚集的ic50为100.57μM。这种新方法是一种快速、廉价的从蛇毒中纯化功能性重组疏水CLPs的方法，对药物治疗的开发很有帮助。
Snake venoms contain a complex mixture of many biological molecules including proteins. The purification of recombinant proteins is a key step in studying their function and structure with affinity chromatography as the common method used in their purification. In bacterial expression systems, hydrophobic recombinant proteins are usually precipitated into inclusion bodies, and contaminants are typically associated with tagged proteins after purification. The purpose of this study was to develop a procedure to purify hydrophobic recombinant proteins without an affinity tag. Snake venom mature C-type lectin-like proteins (CLPs) with a tag were cloned, expressed, and purified by repeated sonication and wash steps. The effects of the signal peptide on the expression and solubility of the recombinant protein were investigated. The CLPs in washed inclusion bodies were solubilized and refolded by dialysis. The CLPs without a tag were successfully purified with a yield 38 times higher than the traditional method, and inhibited blood platelet aggregation with an IC50of 100.57μM in whole blood. This novel procedure is a rapid, and inexpensive method to purify functional recombinant hydrophobic CLPs from snake venoms useful in the development of drug therapies.