Prokaryotic expression and purification of GST-NRP-1 fusion protein
10.3969/j.issn.1000-484X.2015.10.016
- VernacularTitle:GST－NRP－1融合蛋白的原核表达及纯化
- Author:
Zhengxiang HAN
;
Mengjin ZHANG
;
Jie XU
;
Hongmei WANG
;
Xiuping DU
;
Chong CHEN
;
Kailin XU
- Publication Type:Journal Article
- Keywords:
NRP-1;
Prokaryotic expression;
Fusion protein;
Purification
- From:
Chinese Journal of Immunology
2015;(10):1370-1374
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To construct GST-tagged human NRP-1 fusion protein expression vector and induce its expression in Escherichia coli ( E.coli) ,then carry on inclusion body refolding and purification so as to obtain GST-NRP-1 fusion protein.Methods:NRP-1 gene was amplified by RT-PCR and inserted into pCR-blunt vector.Then the reconstructed plasmid was inserted into prokaryotic expression vector pGEX-4T-1.The constructed pGEX-4T-1-NRP-1 expression vector was transformed into BL21 cells and induced by i-sopropyl-β-D-thiogalactoside ( IPTG).Bacterial bodies were disrupted by sonication.Then the soluble fraction of fusion proteins were verified by Western blot and purified by Glutathione Sepharose 4B after inclusion body refolding.Results: The NRP-1 gene fragment was amplified by RT-PCR and inserted into pCR-blunt vector.Fusion protein expression vector pGEX-4T-1-NRP-1 was constructed suc-cessfully.After transformation, GST-NRP-1 expression vector was detected in BL21 cells and obtained purifying protein after refolding.Conclusion:The plasmid GST-NRP-1 was constructed successfully and laid basis for subsequent studies.
