Expression and Purification of Full-Length and Domain-Fragment Recombinant Pentraxin 3 (PTX3) Proteins from Mammalian and Bacterial Cells
Expression and Purification of Full-Length and Domain-Fragment Recombinant Pentraxin 3 (PTX3) Proteins from Mammalian and Bacterial Cells
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来自哺乳动物和细菌细胞的全长和结构域片段重组 Pentraxin 3 (PTX3) 蛋白的表达和纯化
DOI：
10.1007/978-1-0716-0430-4_7
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Hamakubo Takao
中科院分区：
文献类型：
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作者：
Daigo Kenji;Hamakubo Takao
虽然基于细胞的蛋白质表达系统使我们能够获得一定量的适合于后续生物学实验的蛋白质，但在纯化过程中有时会遇到感兴趣的蛋白质的聚集体。五角蛋白3(PTX3)是五角蛋白家族的一员，根据其结构被归类为碳水化合物结合蛋白，它是模式识别受体的体液臂之一，在先天性免疫反应中发挥重要作用。PTX3包括两个结构域：N-末端结构域和C-末端结构域。含有五肽信号的C-末端结构域具有与其他五肽类似的生物学功能，如C-反应蛋白(CRP)和血清淀粉样蛋白-P组分(SAP)。另一方面，N-末端结构域是PTX3所特有的。因此，提供全长或部分片段的PTX3蛋白对于阐明其生物学功能是必不可少的。在此，我们介绍了重组PTX3的表达和纯化。含有精氨酸的缓冲液对于细菌表达的PTX3 N-末端结构域的洗脱至关重要，以最大限度地减少聚集。这种方法可以高产率地纯化全长或结构域片段的重组PTX3蛋白，用于生物学研究。
Although cell-based protein expression systems enable us a certain amount of protein suitable for subsequent biological experiments to be obtained, aggregates of the protein of interest are sometimes encountered during the purification procedure. Pentraxin 3 (PTX3), a member of the pentraxin family that is classified as a carbohydrate-binding protein based on its structure, comprises one of the humoral arms of the pattern recognition receptors that play an important role in the innate immune response. PTX3 comprises two domains; an N-terminal domain and a C-terminal domain. The C-terminal domain containing pentraxin signature has similar biological functions as other pentraxins such as C-reactive protein (CRP) and serum amyloid-P component (SAP). On the other side, the N-terminal domain is specific to PTX3. A supply of the PTX3 protein in full length or partial fragments is thus essential for the elucidation of its biological functions. Here we describe the expression and purification of recombinant PTX3. An arginine-containing buffer is essential for the elution of bacterially expressed PTX3 N-terminal domain to minimize aggregation. This method allows high-yield purification of full-length or domain-fragment recombinant PTX3 proteins for biological study.