Expression and purification of a novel therapeutic single-chain variable fragment antibody against BNP from inclusion bodies of Escherichia coli
Expression and purification of a novel therapeutic single-chain variable fragment antibody against BNP from inclusion bodies of Escherichia coli
复制标题
大肠杆菌包涵体中抗 BNP 的新型治疗性单链可变片段抗体的表达和纯化
DOI：
10.1016/j.pep.2013.10.002
复制
发表时间：
2013-12-01
影响因子：
1.6
通讯作者：
Zhang, Wei
中科院分区：
文献类型：
--
作者：
Bu, Dawei;Zhou, Yuwei;Zhang, Wei
脑钠肽（BNP）分泌异常被认为是脑性盐耗综合征（CSW）的主要发病机制，CSW是由于颅内疾病引起的肾脏钠和水的丢失而导致的低钠血症。规模化制备治疗性单链可变区抗体（scFv）具有中和循环BNP升高的潜在临床应用价值。在本报告中，我们使用最近分离的人源化抗BNP scFv片段（3C 1）作为模型抗体（Ab）来评估这种治疗性蛋白的规模生产的潜力。克隆到pET 22 b（+）中的scFv基因主要以包涵体形式在大肠杆菌中高效表达。coli）Rosetta（DE 3）pLysS细胞。在变性条件下通过Ni-NTA琼脂糖树脂溶解和纯化不溶性片段，并通过含有50 mM Tris-HCl（pH 8.0）、0.15 M NaCl、1 mM EDTA、0.5 M精氨酸、2 mM GSH、1 mM GSSG和5%甘油的有效重折叠缓冲液回收。用PEG 20000浓缩重折叠的scFv片段，并在PBS（含有5%甘油，pH 7.4）中透析。最终产量为每升培养物（3.4g湿重细胞）约10.2mg活性scFv片段。经SDS-PAGE检测，单链抗体纯度大于95%。Western blotting结果显示，重组单链抗体与抗His标签抗体呈免疫反应性。ELISA结果显示，该单链抗体片段与BNP特异性结合，其活性与传统的抗BNP单克隆抗体（mAb）相似。因此，目前的策略允许方便地小规模生产这种治疗性蛋白质。(C)2013 Elsevier Inc. All rights reserved.
Abnormal brain natriuretic peptide (BNP) secretion is regarded as the dominating mechanism of cerebral salt wasting syndrome (CSW), which results from a renal loss of sodium and water during intracranial disease leading to hyponatremia. Scale preparation of therapeutic single-chain variable fragment (scFv) that can neutralize elevated circulating BNP may have potential value for clinical use. In this report, we used a recently isolated humanized anti-BNP scFv fragment (3C1) as model antibody (Ab) to evaluate the potential of scale production of this therapeutic protein. The truncated gene encoding for scFv fragment cloned in pET22b (+) was mainly overexpressed as inclusion bodies in Escherichia coli (E. coli) Rosetta (DE3) pLysS cells. The insoluble fragment was solubilized and purified by Ni-NTA agarose resin under denaturation conditions, and recovered via an effective refolding buffer containing 50 mM Tris-HCl, pH 8.0, 0.15 M NaCl, I mM EDTA, 0.5 M arginine, 2 mM GSH, 1 mM GSSG, and 5% glycerol. The refolded scFv fragment was concentrated by PEG20000, and dialyzed in PBS (containing 5% glycerol, pH 7.4). The final yield was approximately 10.2 mg active scFv fragment per liter of culture (3.4 g wet weight cells). The scFv fragment was more than 95% pure assessed by SDS-PAGE assay. Recombinant scFv fragment with His tag displayed its immunoreactivity with anti-His tag Ab by western blotting. ELISA showed the scFv fragment specifically bound to BNP, and it displayed similar activity as the traditional anti-BNP monoclonal Ab (mAb). Thus, the current strategy allows convenient small-scale production of this therapeutic protein. (C) 2013 Elsevier Inc. All rights reserved.