Plasmid DNA purification via the use of a dual affinity protein.
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 an ethidium bromide agarose gel electrophoresis assay can be used to analyze the eluted pDNA.
Forde
Gareth M
GM
Department of Chemical Engineering, Monash University, Clayton, Victoria, Australia.
D000327:Adsorption; D004247:DNA / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D004996:Ethidium / Q000737:chemistry; D005285:Fermentation; D005982:Glutathione Transferase / Q000737:chemistry; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence
D011993:Recombinant Fusion Proteins; D004247:DNA; D005982:Glutathione Transferase; D004996:Ethidium