LacO-LacI interaction in affinity adsorption of plasmid DNA

Gareth M. Forde, Siddhartha Ghose, Nigel K. H. Slater, Anna V. Hine, Richard A.J. Darby, Anthony G. Hitchcock

Research output: Contribution to journalArticle

Abstract

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. © 2006 Wiley Periodicals, Inc.

Original languageEnglish
Pages (from-to)67-75
Number of pages9
JournalBiotechnology and Bioengineering
Volume95
Issue number1
DOIs
Publication statusPublished - 5 Sep 2006

Keywords

  • affinity chromatography
  • affinity ligands
  • lacO/lacI binding
  • plasmid DNA
  • protein/DNA interaction

Fingerprint Dive into the research topics of 'LacO-LacI interaction in affinity adsorption of plasmid DNA'. Together they form a unique fingerprint.

  • Activities

    • 1 Consultancy

    Consultant, Intrexon Inc.

    Anna V. Hine (Advisor)

    2010

    Activity: Consultancy typesConsultancy

    Cite this

    Forde, G. M., Ghose, S., Slater, N. K. H., Hine, A. V., Darby, R. A. J., & Hitchcock, A. G. (2006). LacO-LacI interaction in affinity adsorption of plasmid DNA. Biotechnology and Bioengineering , 95(1), 67-75. https://doi.org/10.1002/bit.20955