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J Zhejiang Univ (Med Sci)  2019, Vol. 48 Issue (1): 5-11    DOI: 10.3785/j.issn.1008-9292.2019.02.02
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.


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.


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.


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 wordsTransient receptor potential channels      Pyrophosphatases/pharmacology      Recombinant fusion proteins/isolation & purification      Glutathione transferase     
Received: 30 September 2018      Published: 10 May 2019
CLC:  Q71  
Corresponding Authors: YANG Wei     E-mail:;
Cite this article:

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.

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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。



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 
Figure 1 Identification and purification of GST-NUDT9-H fusion protein without detergent
Figure 2 The effect of double concentration of protein inhibitor or the addition of DDM on the extraction of GST-NUDT9-H fusion protein
Figure 3 Re-binding of the lysate with regenerative GST beads leads to higher production of fusion protein
Figure 4 0.025% DDM in wash buffer stabilized the fusion protein in purification system
Figure 5 Optimization on the cleavage time and concentration of thrombin
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