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Nucleic Acids Research, Vol 26, Issue 19 4524-4528, Copyright © 1998 by Oxford University Press

ARTICLES

Purification of plasmids by triplex affinity interaction

T Schluep and CL Cooney
Chemical Engineering Department, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA. thomas.schluep@canji.com

Production of pharmaceutical grade plasmid DNA is an important issue in gene therapy. We developed a method for affinity purification of plasmids by triple helix interaction. This method is based on sequence- specific binding of an oligonucleotide immobilized on a large pore chromatography support to a target sequence on the plasmid. Using design criteria derived from thermodynamic data, we produced a 15mer target sequence which binds strongly to the affinity support under mildly acidic conditions. Plasmid DNA was purified from clarified Escherichia coli lysate by incubation with the affinity beads at pH 5.0 and high NaCl concentration. After extensive washing of the beads, purified plasmid DNA was eluted with alkaline buffer. The purified plasmid showed no RNA or cell DNA contamination in HPLC analysis and total protein concentration was reduced considerably. Due to its mechanical stability and porosity this support can be used in a continuous affinity purification process, which has a high potential for scale up.
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