LacI‐mediated sequence‐specific affinity purification of plasmid DNA for therapeutic applications
| Autor: | Richard A. J. Darby, Anna V. Hine |
|---|---|
| Rok vydání: | 2005 |
| Předmět: |
Recombinant Fusion Proteins
Genetic Vectors Green Fluorescent Proteins Gene Expression Lac repressor Biology Biochemistry chemistry.chemical_compound Plasmid Bacterial Proteins Affinity chromatography Lac Repressors Vaccines DNA Genetics Histidine Molecular Biology Plasmid preparation RNA DNA Genetic Therapy Molecular biology Repressor Proteins genomic DNA Lac Operon chemistry Mutation In vitro recombination Plasmids Biotechnology |
| Zdroj: | The FASEB Journal. 19:1-20 |
| ISSN: | 1530-6860 0892-6638 |
| DOI: | 10.1096/fj.04-2812fje |
| Popis: | Affinity purification of plasmid DNA is an attractive option for the biomanufacture of therapeutic plasmids, which are strictly controlled for levels of host protein, DNA, RNA, and endotoxin. Plasmid vectors are considered to be a safer alternative than viruses for gene therapy, but milligram quantities of DNA are required per dose. Previous affinity approaches have involved triplex DNA formation and a sequence-specific zinc finger protein. We present a more generically applicable protein-based approach, which exploits the lac operator, present in a wide diversity of plasmids, as a target sequence. We used a GFP/His-tagged LacI protein, which is precomplexed with the plasmid, and the resulting complex was immobilized on a solid support (TALON resin). Ensuing elution gives plasmid DNA, in good yield (>80% based on recovered starting material, 35-50% overall process), free from detectable RNA and protein and with minimal genomic DNA contamination. Such an affinity-based process should enhance plasmid purity and ultimately, after appropriate development, may simplify the biomanufacturing process of therapeutic plasmids. |
| Databáze: | OpenAIRE |
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