Isolation and purification of human haptoglobin by ion exchange chromatography
10.7644/j.issn.1674-9960.2016.07.009
- VernacularTitle:离子交换层析分离纯化人结合珠蛋白的研究
- Author:
Jingjing YAN
;
Xiong ZHAO
;
Yuyuan MA
;
Xiaowei MA
;
Jingang ZHANG
- Publication Type:Journal Article
- Keywords:
haptoglobin;
Cohn fraction Ⅳ;
purification;
identification
- From:
Military Medical Sciences
2016;40(7):569-572,592
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop an effective process for isolating and purifying haptoglobin ( Hp) from Cohn fractionⅣby a new ion exchange chromatography and to preliminarily identify and analyze the product of each purification step . Methods The fraction was first diluted and impurities were adsorbed with Rivanol .Then, the supernatant was treated with 50%ammonium sulfate.Finally, the precipitate was redissolved , and Hp was purified further with Q Sepharose Fast Flow chromatography .Native-PAGE was used to measure the activity of the haptoglobin-bound hemoglobin , while SDS-PAGE analysis and immunoblot were used for identification of the target protein .Results After pretreatment , some of the impuri-ties were removed from the Cohn fraction Ⅳ, and the target protein was enriched .In our case, the target protein was Hp and Hp2-2 was the main phenotype in the human plasma fraction Ⅳ.Target protein band and high purity were identified by SDS-PAGE.Immunoblot analysis further proved that this method could successfully isolate the target protein Hp , and the activity of 2.8 U/ml was measured by Native-PAGE method.Conclusion Haptoglobin is successfully isolated from human Cohn fractionⅣwith this method.The purification process is simple and suitable for scale-up production with a good prospect.
