title: Engineering of the LukS-PV and LukF-PV subunits of Staphylococcus aureus Panton-Valentine leukocidin for diagnostic and therapeutic applications.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: School of Molecular Medical Sciences, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2UH, UK. okolie.charles@lmu.edu.ng.
authors.forename: Charles Emeka
authors.initials: CE
authors.lastname: Okolie
authors.forename: Alan
authors.initials: A
authors.lastname: Cockayne
authors.forename: Christopher
authors.initials: C
authors.lastname: Penfold
authors.forename: Richard
authors.initials: R
authors.lastname: James
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001427:Bacterial Toxins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D019295:Computational Biology; D053938:DNA Fragmentation; D004269:DNA, Bacterial / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005098:Exotoxins / Q000235:genetics* / Q000378:metabolism; D007956:Leukocidins / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D013211:Staphylococcus aureus / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004269:DNA, Bacterial; D005098:Exotoxins; D007956:Leukocidins; C078284:Panton-Valentine leukocidin; D011994:Recombinant Proteins