Prokaryotic expression and purification of recombinant CARD9-MBP fusion protein
10.16571/j.cnki.1008-8199.2019.03.004
- VernacularTitle: 胱天蛋白酶募集域蛋白9⁃麦芽糖结合蛋白融合蛋白的原核表达及纯化
- Author:
Dong⁃ling DENG
1
;
Qing⁃tao KONG
1
;
Qing⁃yuan HU
2
;
Hai⁃bo LIU
1
;
Tian⁃biao WEI
2
;
Shan⁃qing HUANG
2
;
Hao CHEN
2
;
Hong SANG
1
Author Information
1. Department of Dermatology, General Hospital of Eastern Theater Command, PLA, Nanjing 210002, Jiangsu,China
2. State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, Jiangsu, China
- Publication Type:Journal Article
- Keywords:
caspase recruitment domain⁃containing protein 9;
prokaryotic expression;
MBP maltose chromatography column;
gel filtration chromatography column
- From:
Journal of Medical Postgraduates
2019;32(3):241-247
- CountryChina
- Language:Chinese
-
Abstract:
Objective CARD9 can activate several pathways involved in immunity, such as NF-ΚB, MAPK, etc. However the mechanism of this process has not yet been elucidated. For conducting relevant experiments in vitro, a prokaryotic expression vector of CARD9-MBP fusion protein has to been construct, and the fusion protein need to be expressed and purified. Methods The coding sequence of CARD9 and MBP genes were amplified by PCR and the recombinant plasmid was correctly inserted into the pET-30a(+) vector. The recombinant plasmid was transformed into E.coli DH5α competent cells and proceeded PCR identification, restriction analysis and gene sequencing. The correct recombinant plasmid was transformed into E.coli BL21(DE3) competent cells. The target protein was induced to express by IPTG under different conditions. Relative molecular weight of the target protein was detected by SDS-PAGE electrophoresis. The CARD9-MBP fusion protein was purified by MBP maltose chromatography column and gel filtration chromatography column, and identificated by MALDI-TOF mass spectrometry after MBP-tag to be removed by HRV3C enzyme. Results The CARD9-MBP fusion protein was successfully constructed and confirmed by PCR and restriction analysis. The result of gene sequencing was consistent with the target sequence. The SDS-PAGE electrophoresis showed that the target protein with molecular mass (MR) about 105 000 was successfully induced to express in E.coli BL21 (DE3). A quite pure CARD9-MBP fusion protein was obtained by purification of MBP maltose chromatography column. Identification by MALDI-TOF mass spectrometry demonstrated that the target protein after MBP-tag to be removed by HRV3C enzyme is CARD9 protein. In the later stage, gel filtration chromatography column was used to obtain further pure CARD9-MBP fusion protein. Conclusion The prokaryotic expression vector of CARD9-MBP fusion protein was successfully constructed and a large number of soluble protein expressed. The purified target protein can be obtained by purification with MBP maltose chromatography column and gel filtration chromatography column.
