Effects of buffer ions on isopropanol-enhanced plasmid purification using Q-Sepharose

Abstract

Ions influence the separation resolution and elution profile obtained by ion-exchange chromatography. This study investigates the effects of buffer ions on the purity and yield of the plasmid purified by Q-Sepharose in a batch adsorption-desorption process when the wash buffers include either 30% or 45% isopropanol. When NaCl, KCl, or MgCl2 was used to remove RNA and proteins, the purified plasmid contained undetectable amounts of proteins and RNA at the salt concentrations that exceeded 1 M or 0.75 M in the presence of 30% and 45% isopropanol, respectively. Using CaCl2 and Na2SO4 led to the incomplete separation of plasmid from RNA. When applied to chromatographic columns packed with 20 or 200 mL of Q-Sepharose to purify plasmid from 300 and 3000 mL bacterial broth, respectively, stepwise elution of various concentrations of isopropanol and MgCl2 yielded a high quantity of plasmid with undetectable amounts of RNA and proteins.