Aqueous-aqueous two-phase systems composed of low molecular weight of polyethylene glycols and dextrans for counter-current chromatographic purification of proteins.
New aqueous-aqueous two-phase systems composed of relatively low molecular weight polymers such as polyethylene glycol (PEG) (Mr: 1000-4000) and dextran (Mr: 10,000 and 40,000) 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 a glucosyltransferase (GTF) from Streptococcus mutans (SM) cell-lysate was successfully demonstrated with a 7.5% PEG 3350-10% dextran T40 system containing 10mM potassium phosphate buffer at pH 9.0. After CCC purification, both PEG and dextran contained in the CCC fractions were easily removed by ultrafiltration in a short period of time. The fractionated column contents containing GTF were analyzed by enzymatic activity as well as sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The recovery of the enzyme from CCC fraction was over 95% as estimated by enzymatic activities.
Shibusawa
Yoichi
Y
Division of Structural Biology and Analytical Science, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan. sibusawa@ps.toyaku.ac.jp <sibusawa@ps.toyaku.ac.jp>
Takeuchi
Naoko
N
Sugawara
Kazusa
K
Yanagida
Akio
A
Shindo
Heisaburo
H
Ito
Yoichiro
Y
D003377:Countercurrent Distribution / Q000295:instrumentation / Q000379:methods*; D003911:Dextrans / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D005964:Glucosyltransferases / Q000032:analysis / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D011092:Polyethylene Glycols / Q000737:chemistry*; D011506:Proteins / Q000032:analysis / Q000302:isolation & purification*
D003911:Dextrans; D011506:Proteins; D011092:Polyethylene Glycols; D005964:Glucosyltransferases