One-step purification of histone deacetylase from Escherichia coli cell-lysate by counter-current chromatography using aqueous two-phase system
- 作者：Yoichi Shibusawa ; Naoko Takeuchi ; Kanako Tsutsumi ; Shigeru Nakano ; Akio Yanagida ; Heisaburo Shindo ; Yoichiro Ito
- 关键词：Aqueous– ; aqueous two-phase systems ; Counter-current chromatography ; Proteins ; Maltose binding protein tagged histone deacetylase
- 刊名：Journal of Chromatography A
- 出版年：2007
- 期刊代码：47_00219673
- 类别：ch
- 出版时间：1 June 2007
- 卷：1151
- 期：1-2
- 页码：158-163
- 文件大小：377 K
摘要
Aqueous–aqueous two-phase (AATP) systems composed of polyethylene glycol (PEG) (molecular mass, Mr:1000–8000) and dextran (Mr:40,000) were evaluated for purification of maltose binding protein tagged-histone deacetylase (MBP-HDAC) by counter-current chromatography (CCC). CCC purification of an MBP-HDAC from Escherichia coli cell-lysate was successfully demonstrated with a 7.0%PEG 3350–10%dextran T40 system containing 10 mM potassium phosphate buffer at pH 9.0. After CCC purification, both polymers in the CCC fractions were easily removed by ultrafiltration in a short period of time. The collected fractions containing target protein were analyzed by an HPLC-based in vitro assay as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis. MBP tag was digested from fusion HDAC during the CCC separation and native HDAC was purified by one-step operation with well preserved deacetyl enzyme activity.