Optimized refolding and characterization of active C-terminal ADAMTS-18 fragment from inclusion bodies of Escherichia coli
Optimized refolding and characterization of active C-terminal ADAMTS-18 fragment from inclusion bodies of Escherichia coli
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DOI：
10.1016/j.pep.2011.11.009
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发表时间：
2012-03-01
影响因子：
1.6
通讯作者：
Zhang, Wei
中科院分区：
文献类型：
--
作者：
Dang, Suying;Hong, Tao;Zhang, Wei
人ADAMTS-18（一种具有血小板反应蛋白1型模块18的崩解素和金属蛋白酶）是ADAMTS家族的新成员。c端ADAMTS-18片段在缺血性脑卒中小鼠模型中具有促进血小板血栓溶解的高效作用，具有显著的临床意义。在本报告中，带GST标签的c端ADAMTS-18片段（命名为rADAMTS-351）在大肠杆菌BL21 (DE3) pLysS中主要以包涵体的形式过表达。不溶性包涵体通过再折叠过程被溶解和重新激活。对rADAMTS-351进行再折叠的最佳缓冲液为50 mM Tris-HCl缓冲液（pH 8.0）、5 mM EDTA、150 mM NaCl、0.1 mM DTT、1 mM GSH和0.2 mM GSSG。将折叠后的rADAMTS-351进行透析，并用谷胱甘肽琼脂糖珠进一步纯化。通过SDS-PAGE和考马斯亮蓝R250染色，最终产品的纯度达到98%。Western blotting显示重组蛋白与抗c -末端ADAMTS-18抗体具有免疫反应性。质谱分析表明分子质量为65,327 Da，与理论预期一致。纯化的rADAMTS-351通过诱导血小板碎片显示其生物活性，与活性c端ADAMTS-18相比，其生物活性范围为81-96%。本研究中描述的表达和重折叠策略可以方便地小规模生产具有生物学功能和治疗潜力的rADAMTS-351。(C) 2011爱思唯尔公司版权所有。
The human ADAMTS-18 (a disintegrin and metalloproteinase with thrombospondin type-1 modules 18) is a new member of the ADAMTS family. The C-terminal ADAMTS-18 fragment is highly effective at promoting platelet thrombus dissolution in murine model of ischemic stroke, showing significant clinical relevance. In this report, the C-terminal ADAMTS-18 fragment with a GST tag (named rADAMTS-351) was overexpressed mainly as inclusion bodies in Escherichia coli BL21 (DE3) pLysS. The insoluble inclusion body was solubilized and reactivated via a refolding procedure. The optimal buffers for refolding rADAMTS-351 was composed of 50 mM Tris-HCl buffer at pH 8.0, 5 mM EDTA, 150 mM NaCl, 0.1 mM DTT, 1 mM GSH, and 0.2 mM GSSG. The refolded rADAMTS-351 was dialyzed and further purified by glutathione-agarose beads. The purity of the final product reached 98% as evaluated by SDS-PAGE and Coomassie Brilliant Blue R250 staining. The recombinant protein displayed its immunoreactivity with anti-C-terminal ADAMTS-18 antibodies by Western blotting. Mass spectroscopic analysis indicated a molecular mass of 65,327 Da as theoretically expected. Purified rADAMTS-351 displayed its bioactivity by inducing platelet fragmentation, which ranged from 81-96% compared to active C-terminal ADAMTS-18 standards. The expression and refolding strategy described in this study allows convenient small-scale production of rADAMTS-351 with biological function and therapeutic potential. (C) 2011 Elsevier Inc. All rights reserved.