LacO-LacI interaction in affinity adsorption of plasmid DNA

Forde, Gareth M., Ghose, Siddhartha, Slater, Nigel K.H., Hine, Anna V., Darby, Richard A.J., & Hitchcock, Anthony G. (2006) LacO-LacI interaction in affinity adsorption of plasmid DNA. Biotechnology and Bioengineering, 95(1), pp. 67-75.

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Current approaches for purifying plasmids from bacterial production systems exploit the physiochemical 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, LacI. Two plasmids were evaluated, the native pUC19 and pUC19 with dual lacO3/lacOs operators (pUC19lacO3/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 LacI peptide that had been immobilized to Streamline™ adsorbent. The KD-values for double stranded DNA (dsDNA) fragments containing lacO1 and lacO3 and lacOs and lacO3 were 5.7 ± 0.3 × 10 -11 M and 4.1 ± 0.2 × 10-11 M respectively, which compare favorably with literature reports of 5 × 10-10 - 1 × 10-9 M for native laCO1 and 1-1.2 × 10-10 M for lacO1 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.

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ID Code: 81572
Item Type: Journal Article
Refereed: Yes
Keywords: Affinity chromatography, Affinity ligands, LacO/lacI binding, Plasmid DNA, Protein/DNA interaction, Amino acids, Bacteria, Binding energy, Cytology, Densitometers, Purification, Densitometric analysis, DNA, DNA fragment, double stranded DNA, protein lacl, repressor protein, unclassified drug, adsorption, article, binding site, controlled study, densitometry, DNA purification, DNA sequence, DNA supercoiling, gel electrophoresis, lactose operon, nonhuman, polymerase chain reaction, protein DNA binding, protein interaction, surface plasmon resonance, Bacterial Proteins, Binding Sites, Chromatography, Affinity, DNA, Bacterial, DNA-Binding Proteins, Lac Operon, Plasmids, Protein Binding, Repressor Proteins, Bacteria (microorganisms)
DOI: 10.1002/bit.20955
ISSN: 0006-3592
Divisions: Current > Schools > School of Chemistry, Physics & Mechanical Engineering
Current > QUT Faculties and Divisions > Science & Engineering Faculty
Copyright Owner: Copyright 2006 Wiley Periodicals, Inc
Deposited On: 05 Feb 2015 22:59
Last Modified: 05 Feb 2015 22:59

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