TY - JOUR

T1 - High-Performance Membrane Chromatography of Supercoiled Plasmid DNA

AU - Giovannini, Roberto

AU - Freitag, Ruth

AU - Tennikova, Tatiana B.

PY - 1998/8/15

Y1 - 1998/8/15

N2 - Membrane adsorbers are well established in protein chromatography. The present paper investigated for the first time the behavior of polynucleotides on these stationary phases, taking a 7.2-kb predominantly supercoiled plasmid as example. Gradient and isocratic elution was studied. In contrast to protein high-performance membrane chromatography (HPMC), isocratic elution is possible in DNA chromatography. In the case of gradient elution, much higher salt concentrations can be used in the starting buffer. Under optimized conditions, both approaches led to a splitting of the single plasmid peak into three maximums, which corresponded to the three-albeit isolated-bands in the agarose gel. Presumably the three fractions were supercoiled, nicked, and open circular plasmid DNA. Linearization of the plasmid lowered the adsorption energy, and the linearized plasmid eluted earlier than the nonlinearized one. The HPMC experiments were compared to similar ones performed using a conventional packed-bed anion-exchange column (Bio-Scale Q2, 7 × 52 mm, 10-μm porous particles) and a novel monolithic-type anion-exchange column (UNO Q1, 7 × 35 mm). The results and characteristic differences observed in these experiments were interpreted in the light of the newly developed theory of HPMC.

AB - Membrane adsorbers are well established in protein chromatography. The present paper investigated for the first time the behavior of polynucleotides on these stationary phases, taking a 7.2-kb predominantly supercoiled plasmid as example. Gradient and isocratic elution was studied. In contrast to protein high-performance membrane chromatography (HPMC), isocratic elution is possible in DNA chromatography. In the case of gradient elution, much higher salt concentrations can be used in the starting buffer. Under optimized conditions, both approaches led to a splitting of the single plasmid peak into three maximums, which corresponded to the three-albeit isolated-bands in the agarose gel. Presumably the three fractions were supercoiled, nicked, and open circular plasmid DNA. Linearization of the plasmid lowered the adsorption energy, and the linearized plasmid eluted earlier than the nonlinearized one. The HPMC experiments were compared to similar ones performed using a conventional packed-bed anion-exchange column (Bio-Scale Q2, 7 × 52 mm, 10-μm porous particles) and a novel monolithic-type anion-exchange column (UNO Q1, 7 × 35 mm). The results and characteristic differences observed in these experiments were interpreted in the light of the newly developed theory of HPMC.

UR - http://www.scopus.com/inward/record.url?scp=0032528715&partnerID=8YFLogxK

U2 - 10.1021/ac980390w

DO - 10.1021/ac980390w

M3 - Article

C2 - 9726161

AN - SCOPUS:0032528715

VL - 70

SP - 3348

EP - 3354

JO - Industrial And Engineering Chemistry Analytical Edition

JF - Industrial And Engineering Chemistry Analytical Edition

SN - 0003-2700

IS - 16

ER -