Engineering of the LukS-PV and LukF-PV subunits of Staphylococcus aureusPanton-Valentine leukocidin for Diagnostic and Therapeutic Applications

BMC Biotechnology

Methodology Article

Alan Cockayne¹  Christopher Penfold¹  Richard James¹  Charles Emeka Okolie² 

[1] School of Molecular Medical Sciences, Centre for Biomolecular Sciences, University of Nottingham, University Park, NG7 2UH, Nottingham, UK;School of Molecular Medical Sciences, Centre for Biomolecular Sciences, University of Nottingham, University Park, NG7 2UH, Nottingham, UK;Department of Biological Sciences, College of Science and Engineering, Landmark University, Omu-Aran, Kwara State, Nigeria;

关键词: Staphylococcus aureus;    Panton-Valentine leukocidin;    Leukocytolytic exotoxin;    Chromatography;    Mass spectrometry;    Immuno-therapy;   

DOI  :  10.1186/1472-6750-13-103

 received in 2013-09-09, accepted in 2013-10-31,  发布年份 2013

来源: Springer

【 摘 要 】

BackgroundStaphylococcus 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.ResultsFollowing 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.ConclusionThe 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.

【 授权许可】

CC BY   
© Okolie et al.; licensee BioMed Central Ltd. 2013