| EPSRC Reference: |
GR/R27716/01 |
| Title: |
Purification of Plasmid Dna For Use In Gene Therapy |
| Principal Investigator: |
Hine, Professor AV |
| Other Investigators: |
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| Researcher Co-Investigators: |
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| Project Partners: |
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| Department: |
Sch of Life and Health Sciences |
| Organisation: |
Aston University |
| Scheme: |
Standard Research (Pre-FEC) |
| Starts: |
10 December 2001 |
Ends: |
09 December 2004 |
Value (£): |
194,955
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| EPSRC Research Topic Classifications: |
| Biological & Medicinal Chem. |
Drug Formulation & Delivery |
| Genomics |
Separation Processes |
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| EPSRC Industrial Sector Classifications: |
| Pharmaceuticals and Biotechnology |
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| Related Grants: |
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| Panel History: |
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Summary on Grant Application Form |
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The proposal aims to employ specific protein-DNA interactions to generate pure plasmid DNA, for use in gene therapy. The work is required because current plasmid purification techniques that are based on anion exchange chromatography result in the co-purification of toxins such as lipopolysaccharide (LPS), from host bacteria. A successful outcome will generate milligram quantities of plasmid DNA, free from such contaminants. To achieve this goal, the lac repressor protein (Lacl) will be exploited. Lac] naturally forms a tetramer which binds to two 20 base pair lac operator sequences. The protein (lacl) may therefore be used as a molecular bridge or 'double-sided sticky-tape'. By transforming the required plasmid into an E. coli strain that over-expresses Lacl, plasmid molecules will become saturated (each plasmid will be bound by one Lacl tetramer), so leaving one DNA-binding site free. By lysing the cells and passing them over a solid support bearing a second lac operator sequence, the second binding site will immobilise the Lacl/plasmid complex, while other components of the cell lysate are washed away. To elute the plasmid DNA, the membrane will be washed with allolactose. Allolactose causes a conformational change in Laci, causing it to release the DNA from both binding sites. Simple size selection will then be used to remove the remaining contaminants: allolactose and the lac repressor protein. The process will have generic application, since many plasmids suitable for use in gene therapy contain the lac operator sequence. The proposal is supported by preliminary data in which a zinc finger protein has been used to purify plasmid DNA from bacterial cells.
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Key Findings |
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This information can now be found on Gateway to Research (GtR) http://gtr.rcuk.ac.uk
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Potential use in non-academic contexts |
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This information can now be found on Gateway to Research (GtR) http://gtr.rcuk.ac.uk
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Impacts |
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| Description |
This information can now be found on Gateway to Research (GtR) http://gtr.rcuk.ac.uk |
| Summary |
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| Date Materialised |
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Sectors submitted by the Researcher |
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This information can now be found on Gateway to Research (GtR) http://gtr.rcuk.ac.uk
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| Project URL: |
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| Further Information: |
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| Organisation Website: |
http://www.aston.ac.uk |