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| Extraction and purification of NUDT9 homology domain of human transient receptor potential melastatin 2 channel |
YE Peiwu1( ),YU Xiafei1,MA Cheng2,YANG Wei1() |
1.Department of Biophysics, Zhejiang University School of Medicine, Hangzhou 310058, China
2.Protein Facility, Zhejiang University School of Medicine, Hangzhou 310058, China |
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Abstract Objective To develop methods of extraction and purification of C-terminal NUDT9 homology domain of human transient receptor potential melastatin 2 (TRPM2) channel. Methods After sonication and centrifuge of Escherichia coli strain Rosetta (DE3) which was induced by isopropylthio-β-D-galactoside, GST-NUDT9-H was collected after the binding of supernatant with GST beads and eluted with reduced glutathione. Then the elution buffer containing fusion protein was purified by size exclusion chromatography after concentration and centrifuge. Finally, with the cleavage of thrombin and binding with the GST beads, NUDT9-H with high purity in supernatant was collected. Results The GST-NUDT9-H fusion protein was stabilized with lysis buffer containing 0.5% n-dodecyl-β-d-maltoside (DDM), and wash buffer containing 0.025% DDM in size-exclusion chromatography system, and finally the NUDT9-H with high purity was obtained after cleaved by thrombin (1 U/2 mg fusion protein) for 24 h. Conclusion Due to the poor stability of NUDT9-H, it is necessary to add DDM in extraction and purification buffer to stabilize the conformation of NUDT9-H, so as to increase its yields and purity.
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Received: 30 September 2018
Published: 10 May 2019
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Corresponding Authors:
YANG Wei
E-mail: yegucheng@zju.edu.cn;yangwei@zju.edu.cn
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人源瞬时受体电位M2型通道Nudix水解酶9同源结构域的提取和纯化
目的 摸索人源瞬时受体电位M2型通道（TRPM2）羧基端Nudix水解酶9（NUDT9）同源结构域（NUDT9-H）的蛋白提取和纯化方法。 方法 异丙基硫代半乳糖苷诱导表达的Rosetta（DE3）大肠埃希菌菌株经超声破碎和离心后，将收集到的上清液与GST磁珠结合并用还原型谷胱甘肽洗脱以抽提GST-NUDT9-H融合蛋白，浓缩离心后再经分子排阻色谱层析纯化，最后经凝血酶酶切并与GST磁珠结合即得NUDT9-H蛋白。 结果 含有0.5%的十二烷基-β-D-麦芽糖苷（DDM）裂解溶液体系和0.025%的DDM分子排阻层析色谱溶液体系能够提高GST-NUDT9-H融合蛋白的稳定性，再按每2 mg融合蛋白加入1 U凝血酶切割24 h，即获得高纯度NUDT9-H蛋白。 结论 NUDT9-H蛋白稳定性较差，提取和纯化体系中需添加DDM以稳定其构象从而提高目的蛋白的产量和纯度。
关键词：
瞬时受体电位通道,
焦磷酸酶类/药理学,
重组融合蛋白质类/分离和提纯,
谷胱甘肽转移酶
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