OKOLIE, CE and Cockayne, Alan and Penfold, Christopher and James, Richard (2013) Engineering of the LukS-PV and LukF-PV subunits of Staphylococcus aureus Panton-Valentine leukocidin for Diagnostic and Therapeutic Applications. BMC Biotechnology, 13. pp. 103-115.
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Engineering of the LukS-PV and LukF-PV OKOLIE 2013.pdf Download (1MB) | Preview |
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Engineering of the LukS-PV and LukF-PV OKOLIE 2013.pdf Download (1MB) | Preview |
Abstract
Abstract Background: Staphylococcus aureus produces several toxins, including Panton-Valentine leukocidin (PVL). The involvement of PVL in primary skin infections, necrotizing pneumonia, musculoskeletal disorders, brain abscess, and other diseases, some of which are life-threatening, has been reported. Following expert opinion, we aimed to provide the tools for establishment of sequence-based diagnostics and therapeutics for those conditions. We engineered the synergistic S and F (LukS-PV and LukF-PV respectively) pro-toxin subunits from Staphylococcus aureus USA400 into separate expression E. coli BL21(DE3)-pLysS hosts. Results: Following Nickel affinity chromatography (NAC), the F subunit came out without bands of impurity. The S sub-unit did not come off very pure after NAC thus necessitating further purification by size exclusion and ion-exchange chromatography. The purification plots showed that the BioLogic-LP and AKTA systems are reliable for following the progress of the chromatographic purification in real-time. Computer predicted Mw for the 6His-LukF-PV and 6His-LukS-PV were 35645.41 Da and 33530.04 Da respectively, while the mass spectrometry results were 35643.57 Da and 33528.34 Da respectively. Conclusion: The BioLogic-LP and AKTA systems are commendable for reliability and user-friendliness. As a recent work elsewhere also reported that a second round of chromatography was necessary to purify the S subunit after the first attempt, we speculate that the S subunit might contain yet unidentified motif(s) requiring further treatment. The purified S and F sub-units of PVL were supplied to the Nottingham Cancer Immunotherapy group who used them to establish sequence-based monoclonal antibodies for diagnostic and therapeutic uses targeting PVL.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | Staphylococcus aureus, Panton-Valentine leukocidin, Leukocytolytic exotoxin, Chromatography, Mass spectrometry, Immuno-therapy |
| Subjects: | Q Science > Q Science (General) Q Science > QR Microbiology T Technology > T Technology (General) |
| Divisions: | Faculty of Medicine, Health and Life Sciences > School of Biological Sciences |
| Depositing User: | DR. CHARLES OKOLIE |
| Date Deposited: | 29 Feb 2016 12:27 |
| Last Modified: | 13 Sep 2019 15:25 |
| URI: | https://eprints.lmu.edu.ng/id/eprint/398 |
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