1994 Fiscal Year Final Research Report Summary
Separation of Biopolymers by Countercurrent Chromatography
| Project/Area Number |
05671792
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Physical pharmacy
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| Research Institution | Tokyo University of Pharmacy and Life Science |
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Principal Investigator |
SHIBUSAWA Yoichi Tokyo University of Pharmacy and Life Science, School of Pharmacy, Lecturer, 薬学部, 講師 (10102708)
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| Project Period (FY) |
1993 – 1994
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| Keywords | Countercurrent Chromatography / Human Serum Lipoproteins / Aqueous Polymer Two Phase Systems / DNA Binding Proteins |
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| Research Abstract |
Three main classes lipoproteins from human serum, one step purification of single stranded binding protein (SSB) and H-NA from E.coli were studied by countercurrent chromatography.
High- and low-density lipoproteins (HDLs, LDLs) were fractionated by countercurrent chromatography using the cross-axis coil planet centrifuge using two-phase aqueous polymer systems. The best separations were achieved with a polymer phase system composed of 16% polyethylene glycol 1000 and 12.5% dibasic potassium phosphate at pH 9.4.HDL-LDL fractions were collected from 4 ml of human serum within 3h. Very-low-lipoproteins (VLDLs) were collected together with serum proteins. Each fraction was concentrated and subjected to hydroxyapatite chromatography to obtain three lipoprotein fractions (HDLs, LDLs and VLDLs) all free of serum proteins, such as albumin and globulin. Each lipoprotein was confirmed by agarose gel electrophoresis.
SSB purification from crude extracts of E.coli was performed by cross-axis coil planet centrifuge using aqueous polymer-two phase system composed of 16% PEG 1000-17% ammonium sulfate. Countercurrent chromatogram gives two main peaks, one contains most of proteins produced by E.coli, and the other one (fraction 51) contains SSB.SSB was confirmed by SDS-PAGE.
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