One-step purification of histone deacetylase from Escherichia coli cell-lysate by counter-current chromatography using aqueous two-phase system
One-step purification of histone deacetylase from Escherichia coli cell-lysate by counter-current chromatography using aqueous two-phase system
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DOI：
10.1016/j.chroma.2007.01.111
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发表时间：
2007-06-01
影响因子：
4.1
通讯作者：
Ito, Yoichiro
中科院分区：
文献类型：
--
作者：
Shibusawa, Yoichi;Takeuchi, Naoko;Ito, Yoichiro
研究了由聚乙二醇（PEG）（分子量：1000-8000）和葡聚糖（分子量：40,000）组成的水-水两相（AATP）体系对麦芽糖结合蛋白标记组蛋白脱乙酰酶（MBP-HDAC）的逆流色谱纯化。用含有10 mM磷酸钾缓冲液（pH 9.0）的7.0% PEG 3350-10%葡聚糖T40系统成功地证明了从大肠杆菌细胞裂解物中CCC纯化MBP-HDAC。CCC纯化后，CCC组分中的两种聚合物都很容易在短时间内通过超滤除去。通过基于HPLC的体外测定以及十二烷基硫酸钠聚丙烯酰胺凝胶电泳分析收集的含有靶蛋白的级分。在CCC分离过程中从融合HDAC中消化MBP标签，并通过一步操作纯化天然HDAC，其脱乙酰酶活性保留良好。(C)2007 Elsevier B. V.保留所有权利。
Aqueous-aqueous two-phase (AATP) systems composed of polyethylene glycol (PEG) (molecular mass, M-r:1000-8000) and dextran (M-r: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. (C) 2007 Elsevier B.V. All rights reserved.