Studies on the adsorption of cell impurities from plasmid-containing lysates to phenyl boronic acid chromatographic beads.
Plasmid DNA (pDNA) is purified directly from alkaline lysis-derived Escherichia coli (E. coli) lysates by phenyl boronate (PB) chromatography. The method explores the ability of PB ligands to bind covalently, but reversibly, to cis-diol-containing impurities like RNA and lipopolysaccharides (LPS), leaving pDNA in solution. In spite of this specificity, cis-diol free species like proteins and genomic DNA (gDNA) are also removed. This is a major advantage since the process is designed to keep the target pDNA from binding. The focus of this paper is on the study of the secondary interactions between the impurities (RNA, gDNA, proteins, LPS) in a pDNA-containing lysate and 3-amino PB controlled pore glass (CPG) matrices. Runs were designed to evaluate the role of adsorption buffer composition, feed type (pH, salt content), CPG matrix and sample pretreatment (RNase A, isopropanol precipitation). Water was chosen as the adsorption buffer over MgCl(2) solutions since it maximised pDNA yield (96.2±4.9%) and protein removal (61.3±3.0%), while providing for a substantial removal of RNA (65.5±3.5%) and gDNA (44.7±14.1%). Although the use of pH 3.5 maximised removal of impurities (~75%), the best compromise between plasmid yield (~96%) and RNA clearance (~60-70%) was obtained for a pH of 5.2. Plasmid yield was maximal (>96%) when the concentration of acetate and potassium ions in the incoming lysate feed were 1.7 M and 1.0 M, respectively. The pre-treatment of lysates with RNase A deteriorated the performance since the resulting oligoribonucleotides lack the cis-diol group at their 3' termini. Overall, the results support the idea that charge transfer interactions between the boron atom at acidic pH and electron donor groups in the aromatic bases of nucleic acids and side residues of proteins are responsible for the non-specific removal of gDNA, RNA and proteins.
IBB-Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Lisboa, Portugal.
A Gabriela
AG
Gomes
Ana M
AM
Azevedo
M Raquel
MR
Aires-Barros
D Miguel F
DM
Prazeres
D000327:Adsorption; D001897:Boronic Acids / Q000737:chemistry*; D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000379:methods*; D004247:DNA / Q000302:isolation & purification; D005898:Glass; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D010957:Plasmids / Q000302:isolation & purification*
D001897:Boronic Acids; C028592:3-aminobenzeneboronic acid; D004247:DNA