Current approaches for purifying plasmids from bacterial production systems exploit the physio-chemical properties of nucleic acids in non-specific capture systems. In this study, an affinity system for plasmid DNA (pDNA) purification has been developed utilizing the interaction between the lac operon (lacO) sequence contained in the pDNA and a 64mer synthetic peptide representing the DNA-binding domain of the lac repressor protein, Lacl. Two plasmids were evaluated, the native pUC19 and pUC19 with dual lacO3/lacOs operators (pUC19(lacO3)/(lacOs)), where the lacOs operator is perfectly symmetrical. The DNA-protein affinity interaction was evaluated by surface plasmon resonance using a Biacore system. The affinity capture of DNA in a chromatography system was evaluated using Lacl peptide that had been immobilized to Streamline m adsorbent. The K-D-values for double stranded DNA (dsDNA) fragments containing lacO(1) and lacO(3) and lacOs and lacO(3) were 5.7 +/- 0.3 x 10(-11) M and 4.1 +/- 0.2 x 10(-11) M respectively, which compare favorably with literature reports of 5 x 10(-10)-1 x 10(-9) M for native lacO(1) and 1-1.2 x 10(-10) M for lacO(1) in a saline buffer. Densitometric analysis of the gel bands from the affinity chromatography run clearly showed a significant preference for capture of the supercoiled fraction from the feed pDNA sample. The results indicate the feasibility of the affinity approach for pDNA capture and purification using native protein-DNA interaction. (c) 2006 Wiley Periodicals, Inc.