AI Article Synopsis
- Aqueous-aqueous two-phase (AATP) systems using polyethylene glycol (PEG) and dextran were tested for purifying maltose binding protein tagged-histone deacetylase (MBP-HDAC) through counter-current chromatography (CCC).
- The MBP-HDAC was successfully purified from Escherichia coli cell-lysate using a solution containing 7.0% PEG 3350 and 10% dextran T40, along with potassium phosphate buffer at pH 9.0.
- After CCC purification, the polymers were easily removed from the protein fractions, and the native HDAC maintained its deacetylase activity after the MBP tag was digested during the separation process.
Article Abstract
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.
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| http://dx.doi.org/10.1016/j.chroma.2007.01.111 | DOI Listing |
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