Extraction and purification of NUDT9 homology domain of human transient receptor potential melastatin 2 channel.
- Author:
Peiwu YE
1
;
Xiafei YU
1
;
Cheng MA
2
;
Wei YANG
1
Author Information
1. Department of Biophysics, Zhejiang University School of Medicine, Hangzhou 310058, China.
2. Protein Facility, Zhejiang University School of Medicine, Hangzhou 310058, China.
- Publication Type:Journal Article
- MeSH:
Escherichia coli;
genetics;
Glucosides;
chemistry;
Humans;
Protein Domains;
Protein Stability;
Pyrophosphatases;
chemistry;
genetics;
isolation & purification;
Recombinant Fusion Proteins;
chemistry;
isolation & purification;
TRPM Cation Channels;
chemistry;
isolation & purification;
Thrombin;
metabolism
- From:
Journal of Zhejiang University. Medical sciences
2019;48(1):5-11
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE:To develop methods of extraction and purification of Cterminal NUDT9 homology domain of human transient receptor potential melastatin 2 (TRPM2) channel.
METHODS:After sonication and centrifuge of 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.
CONCLUSIONS: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.
