SpringerProtocols: Abstract: Plasmid DNA Purification Via the Use of a Dual Affinity Protein

18. Plasmid DNA Purification Via the Use of a Dual Affinity Protein

By: Gareth M. Forde²

Abstract

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Methods are presented for the production, affinity purification and analysis of plasmid DNA (pDNA). Batch fermentation is used for the production of the pDNA, and expanded bed chromatography, via the use of a dual affinity glutathione S-transferase (GST) fusion protein, is used for the capture and purification of the pDNA. The protein is composed of GST, which displays affinity for glutathione immobilized to a solid-phase adsorbent, fused to a zinc finger transcription factor, which displays affinity for a target 9-base pair sequence contained within the target pDNA. A Picogreen™ fluorescence assay and/or anx ethidium bromide agarose gel electrophoresis assay can be used to analyze the eluted pDNA.

Affiliation(s): (2) BioMarin Pharmaceutical Inc., CA

Book Title: Affinity Chromatography: Methods and Protocols

Series: Methods in Molecular Biology | Volume: 421 | Pub. Date: Nov-30-2007 | Page Range: 275-284 | DOI: 10.1007/978-1-59745-582-4_18

Subject: Cell Biology

Key Words: Plasmid DNA - affinity purification - fermentation - chromatography - expanded bed adsorption

References

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1.	Legendre JY, Haensler J, Remy JS. Non-viral gene delivery systems (1996). Médecine/Sciences 12, 1334–1341.

2.	Ferreira GNM, Monteiro GA, Prazeres DMF, Cabral JMS. Downstream processing of plasmid DNA for gene therapy and DNA vaccine applications (2000). Trends Biotechnol. 18, 380–388.

3.	Scherman D. Towards non viral gene therapy (2001). Bull. Acad. Natl. Med. 185, 1683–1697.

4.	Davis HL. Plasmid DNA expression systems for the purpose of immunization (1997). Curr. Opin. Biotechnol. 8, 635–640.

5.	Levy MS, O’Kennedy RD, Ayazi-Shamlou P, Dunnill P. Biochemical engineering approaches to the challenges of producing pure plasmid DNA (2001). Trends Biotechnol. 18, 296–305.

6.	Diogo MM, Ribeiro SC, Queiroz JA, Monteiro GA, Tordo N, Perrin P, Prazeres DMF. Production, purification and analysis of an experimental DNA vaccine against rabies (2001). J. Gene Med. 3, 577–584.

7.	Johansen P, Raynaud C, Yang M, Colston MJ, Tascon RE, Lowrie DB. Anti-mycobacterial immunity induced by a single injection of M. leprae Hsp65-encoding plasmid DNA in biodegradable microspheres (2003). Immunol. Lett. 90, 81–85.

8.	Bouchie A. DNA vaccine deployed for endangered condors (2003). Nat. Biotechnol. 21, 11.

9.	Varley DL, Hitchcock AG, Weiss AME, Horler WA, Cowell R, Peddie L, Sharpe GS, Thatcher DR, Hanak JAJ. Production of plasmid DNA for human gene therapy using modified alkaline cell lysis and expanded bed anion exchange chromatography (1999). Bioseparation 8, 209–217.

10.	Chase HA. The use of affinity adsorbents in expanded bed adsorption (1998). J. Mol. Recognit. 11, 217–221.

11.	Molecular Probes™ Quant-iT™ PicoGreen™ dsDNA Reagent and Kits (2005), Product Information: MP07581.

12.	Sambrook JS, Russell DW. Molecular Cloning: A Laboratory Manual, Third Edition, 2001, CSHL Press, Cold Spring Harbor, New York.

13.	Matt T, Martinez-Yamout MA, Dyson HJ, Wright PE. The CBP/p300 TAZ1 domain in its native state is not a binding partner of MDM2 (2004). Biochem. J. 381, 685–691.

14.	Amersham Biosciences™, Glutathione Sepharose 4 Fast Flow™, Affinity Chromatography 2003, Data File 18-1174-85 AA: 1–8.

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