| Project/Area Number |
17590042
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Physical pharmacy
|
|---|
| Research Institution | Tokyo University of Pharmacy and Life Science |
|---|
Principal Investigator |
SHIBUSAWA Yoichi Tokyo University of Pharmacy and Life Sciences, School of Pharmacy, Associate Professor, 薬学部, 助教授 (10102708)
|
|---|
| Project Period (FY) |
2005 – 2006
|
| Project Status |
Completed (Fiscal Year 2006)
|
| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2006: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2005: ¥1,700,000 (Direct Cost: ¥1,700,000)
|
|---|
| Keywords | Recombinant enzyme / Counter-current chromatography / One-step purification / Analyses of function |
|---|
| Research Abstract |
New aqueous-aqueous two-phase (AATP) systems composed of relatively low molecular weight polymers such as polyethylene glycol (PEG) and dextran were evaluated for purification of proteins by counter-current chromatography (CCC). The compositions of aqueous two-phase systems were optimized by measuring parameters such as viscosity and volume ratio between the two phases. CCC purification of glucosyltransferase (GTF) from Streptococcus mutants (SM) cell-lysate was successfully demonstrated with a 7.5% PEG 3350-10% dextran T40 system containing 10 mM potassium phosphate buffer at pH 9.0.
AATP were also evaluated for purification of maltose binding protein tagged-histone deacetylase (MBP-HDAC) by 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 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.
|
