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浙江大学学报（医学版）
浙江大学学报（医学版）  2019, Vol. 48 Issue (1): 5-11    DOI: 10.3785/j.issn.1008-9292.2019.02.02
专题报道     
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
叶培武1()
1. 浙江大学医学院生物物理系， 浙江 杭州 310058
2. 浙江大学医学院蛋白质平台， 浙江 杭州 310058
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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摘要： 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    
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.
Key words: Transient receptor potential channels    Pyrophosphatases/pharmacology    Recombinant fusion proteins/isolation & purification    Glutathione transferase
收稿日期: 2018-09-30 出版日期: 2019-05-10
:  Q71  
基金资助: 国家自然科学基金(31872796);浙江省自然科学基金杰出青年科学基金项目(LR16H090001)
作者简介: 叶培武（1995—），男，硕士研究生，主要从事蛋白质纯化及线虫神经元研究；E-mail： yegucheng@zju.edu.cn； https://orcid.org/0000-0002-9177-4022|杨　巍（1976—），男，博士，教授，博士生导师，主要从事离子通道研究；E-mail： yangwei@zju.edu.cn； https://orcid.org/0000-0003-3065-1843
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引用本文:

叶培武. 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[J]. 浙江大学学报（医学版）, 2019, 48(1): 5-11.

YE Peiwu,YU Xiafei,MA Cheng,YANG Wei. Extraction and purification of NUDT9 homology domain of human transient receptor potential melastatin 2 channel. J Zhejiang Univ (Med Sci), 2019, 48(1): 5-11.

链接本文:

http://www.zjujournals.com/med/CN/10.3785/j.issn.1008-9292.2019.02.02    或    http://www.zjujournals.com/med/CN/Y2019/V48/I1/5

图1  未添加表面活性剂GST-NUDT9-H融合蛋白的鉴定和纯化
图2  加倍蛋白酶抑制剂用量或添加DDM对NUDT9-H融合蛋白提取的影响
图3  细菌裂解上清液与GST磁珠重结合可以提高产量
图4  在纯化缓冲液体系中添加0.025%DDM有助于获得稳定的目的蛋白
图5  融合蛋白酶切时间和浓度优化
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