Purification of single-strand DNA binding protein from an Escherichia coli lysate using counter-current chromatography, partition and precipitation.
Single-strand DNA binding protein (SSB) from Escherichia coli lysate was purified by counter-current chromatography (CCC) using the ammonium sulfate precipitation method in a coiled column. About 5 ml of E. coli lysate was separated by CCC using a polymer phase system composed of 16% (w/w) polyethylene glycol (PEG) 1000 and 17% (w/w) ammonium sulfate aqueous polymer two-phase solvent system. The precipitation of proteins in the lysate took place in the CCC column, and the SSB protein was eluted in the fraction 51-56. Many other impurities were either eluted immediately after the solvent front or precipitated in the column. The identities of the proteins in the fractions and in the precipitate were confirmed by SDS-polyacrylamide gel electrophoresis with Coomassie Brilliant Blue staining.
Shibusawa
Yoichi
Y
Department of Analytical Chemistry, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan. sibusawa@ps.toyaku.ac.jp
Ino
Yohko
Y
Kinebuchi
Takashi
T
Shimizu
Mitsuhiro
M
Shindo
Heisaburo
H
Ito
Yoichiro
Y
D011232:Chemical Precipitation; D003377:Countercurrent Distribution / Q000379:methods*; D004268:DNA-Binding Proteins / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000302:isolation & purification*
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C476430:SSB protein, E coli