Preparative fractionation of protein, RNA, and plasmid DNA using centrifugal precipitation chromatography with tubular dialysis membrane inside a convoluted tubing as separation channel.
Fractionation of clarified E. coli lysate components in bench-scale and preparative-scale centrifugal precipitation chromatography (CPC), using a solution of cationic surfactant cetyltrimethylammonium bromide (CTAB) containing 0.5 M NaCl as precipitant, are compared here. Step gradient of CTAB from 0.50% to 0.16% (w/v) gave a successful fractionation in bench-scale CPC; however, a linear gradient of lower CTAB concentration, 0.20-0% (w/v), was used in the preparative scale and resulted in similar fractionation. The preparative-scale CPC has a superior sample loading capacity by the use of tubular dialysis membrane inside convoluted tubing as the separation channel. In this study, the quantity of the sample loaded into the preparative CPC was about 15 times more than that in the bench scale, and in a single run the preparative CPC could prepare approximately 3 mg of plasmid DNA with about 96% of RNA removed. The higher surface area per length of the separation channel in the preparative CPC was believed to benefit mass transfer of CTAB across the membrane, leading to less CTAB being required in the process.
Tomanee
Panarat
P
Department of Chemical Engineering, Biopharmaceutical Technology Institute, Lehigh University, Bethlehem, Pennsylvania 18015, USA.
Hsu
James T
JT
Ito
Yoichiro
Y
D002498:Centrifugation / Q000295:instrumentation / Q000379:methods*; D002845:Chromatography / Q000295:instrumentation / Q000379:methods*; D004247:DNA / Q000302:isolation & purification*; D003956:Dialysis / Q000295:instrumentation / Q000379:methods*; D004926:Escherichia coli; D010957:Plasmids / Q000302:isolation & purification*; D011506:Proteins / Q000302:isolation & purification*; D012313:RNA / Q000302:isolation & purification*
D011506:Proteins; D012313:RNA; D004247:DNA