Bu Dawei, Zhou Yuwei, Tang Jian, Jing Fang, Zhang Wei
Department of Pharmacology, Institutes for Advanced Studies in Multidisciplinary Science and Technology, Shanghai 200062, PR China.
Protein Expr Purif. 2013 Dec;92(2):203-7. doi: 10.1016/j.pep.2013.10.002. Epub 2013 Oct 12.
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, 1 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.
异常的脑钠肽（BNP）分泌被认为是脑性盐耗综合征（CSW）的主要发病机制，CSW是由颅内疾病期间肾脏失钠和失水导致低钠血症引起的。制备能够中和循环中升高的BNP的治疗性单链可变片段（scFv）可能具有临床应用价值。在本报告中，我们使用最近分离的人源化抗BNP scFv片段（3C1）作为模型抗体（Ab）来评估这种治疗性蛋白质的规模化生产潜力。克隆到pET22b（+）中的编码scFv片段的截短基因在大肠杆菌（E. coli）Rosetta（DE3）pLysS细胞中主要以包涵体形式过量表达。不溶性片段在变性条件下通过Ni-NTA琼脂糖树脂溶解和纯化，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%甘油的有效复性缓冲液复性。复性后的scFv片段通过PEG20000浓缩，并在PBS（含5%甘油，pH 7.4）中透析。最终产量约为每升培养物10.2 mg活性scFv片段（3.4 g湿重细胞）。通过SDS-PAGE分析评估，scFv片段的纯度超过95%。带有His标签的重组scFv片段通过蛋白质免疫印迹法显示与抗His标签抗体具有免疫反应性。酶联免疫吸附测定（ELISA）表明scFv片段与BNP特异性结合，并且其活性与传统抗BNP单克隆抗体（mAb）相似。因此，当前策略允许方便地小规模生产这种治疗性蛋白质。
