High-level expression and efficient purification of recombinant human long pentraxin PTX3 in Chinese hamster ovary cells
High-level expression and efficient purification of recombinant human long pentraxin PTX3 in Chinese hamster ovary cells
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DOI：
10.1016/j.pep.2006.07.009
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发表时间：
2007-01-01
影响因子：
1.6
通讯作者：
Verdoliva, Antonio
中科院分区：
文献类型：
--
作者：
Rivieccio, Vincenzo;Esposito, Adelaide;Verdoliva, Antonio
PTX3是一种分泌型多聚体糖蛋白，通过对C1q亚基的特异性识别激活经典的补体途径，在天然免疫中发挥关键作用。介绍了一种在中国仓鼠卵巢细胞(CHO)中高水平表达重组人PTX3的方法，该细胞适合在装有无血清化学限定培养基的旋转培养瓶中悬浮生长，并产生约50 mg PTX3/L的培养物。本文还报道了用阴离子交换、羟基磷灰石和尺寸排斥层析等方法从上清液中提纯蛋白质的方法。此三步法可使LIS获得回收率接近70%、纯度超过95%、最终宿主细胞蛋白(HCP)含量低于150ppm的PTX3。经C1q结合的EL ISA分析表明，重组纯化的PTX3保持了其生物学活性；圆二色谱、荧光分析、天然和SDS-PAGE实验表明，PTX3呈现出一种复杂的四级结构，其二级结构含量高，与人的短五肽C反应蛋白(CRP)和血清淀粉样蛋白P组分(SAP)大不相同。(C)2006 Elsevier Inc.保留所有权利。
PTX3 is a secreted multimeric glycoprotein which plays a key role in innate immunity by activating the classical complement pathway through specific recognition of the C1q Subunit. A method is described for the high level expression of the recombinant human PTX3 in Chinese hamster ovary cells (CHO), adapted to a Suspension growth in spinner flasks containing a serum-free chemically defined medium and producing about 50 mg of PTX3/L Of Culture. A purification procedure to produce a homogeneous protein preparation from the supernatant, by means of anion exchange, hydroxyapatite and size exclusion chromatography, is also reported. This three-step protocol allows Lis to obtain PTX3 with a recovery yield close to 70%, a purity degree exceeding 95%, and a final host cell protein (HCP) content lower than 150ppm. The recombinant purified PTX3 retains its biological activity, as demonstrated by C1q binding ELISA assay, and displays a complex quaternary structure characterized by a high secondary structure content quite different from human short pentraxin C-reactive protein (CRP) and serum amyloid P component (SAP), as determined by Circular dichroism, fluorescence analysis, and native and SDS-PAGE experiments. (c) 2006 Elsevier Inc. All rights reserved.