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.

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.

Figure 1

Start-up amplification and restriction digests of rlukS-PV and rlukF-PV. A. PCR amplification of rlukS-PV and rlukF-PV from S. aureus MW2 template genomic DNA. Lane L, 100 bp DNA Marker (NEB, UK); Lanes 1 and 2, rlukS-PV, 868 bp; Lane 3, Molecular grade water used as PCR negative control; Lanes 4 and 5 rlukF-PV, 919 bp. B. Double digests (NcoI and XhoI) of minipreps of the intermediate host E. coli 5α-pGEMT-Easy-luk-PV to release the insert DNA fragments. Lane 1, Undigested pGEMT-Easy-rlukF-PV; Lane 2, Digested pGEMT-Easy-rlukF-PV showing the cleaved insert rlukF-PV below the 1.0 kb mark; Lane 3, Duplicate of lane 1; Lane 4, Duplicate of lane 2; Lane 5, Undigested pGEMT-Easy-rlukS-PV; Lane 6, Digested pGEMT-Easy-rlukS-PV showing the cleaved insert rlukS-PV, below the 1.0 kb mark; Lane 7, Duplicate of lane 5; Lane 8, Duplicate of lane 6; Lane 9, Undigested pET expression vector; Lane 10, pET vector cut with NcoI and XhoI (the next home of the cleaved insert DNA fragments); Lane 11, Molecular grade water; Lane L, 1 kb DNA Marker (NEB, UK).

Figure 2

Reconstruction and amplification of rlukS-PV and rlukF-PVfrom protein expression system. A. Gene map of pET-21d(+)-lukF-PV (Reconstructed using VECTOR NTI software). The insert (rlukF-PV) locates within the NcoI and AvaI RDE sites, technically between the T7 promoter and terminator, all confirmed by sequencing. NOTE: The VECTOR NTI prefers the non-specific nuclease (AvaI), which recognises the degenerate sequence (CYCGRG), over the specific XhoI used in the cloning experiments which specifically recognises the sequence (CTCGAG) engineered into the primers used for recombination. B. Amplification of rlukS-PV and rlukF-PV from the expression system. Lane 1, rlukS-PV amplified from expression E. coli BL21(DE3)-pET-21d(+)-lukS-PV; Lane 2, rlukF-PV amplified from expression E. coli BL21(DE3)-pET-21d(+)-lukF-PV; Lane 3, Molecular grade water used as negative PCR control; Lane 4, 100 bp DNA ladder.

Figure 3

Expression and Ni⁺⁺-affinity purification of S and F protein subunits. A. Over-expression of fusion LukS-PV and LukF-PV (10% SDS-PAGE) Lane 1, Protein Mw standard; Lanes 2 & 3, Total soluble lysate of E. coli BL21(DE3)pLysS-pET-21d(+)-lukF-PV after induction with IPTG; Lanes 4 & 5, Total soluble lysate of E. coli BL21(DE3)pLysS-pET-21d(+)-lukS-PV after induction with IPTG; Lane 6, Uninduced E. coli BL21(DE3)-pET-21d(+)-lukS-PV; Lane 7, Un induced E. coli BL21(DE3)-pET-21d(+)-lukF-PV. 3B and C. Purity of His-Tagged LukS-PV and LukF-PV following Nickel Affinity chromatography. B. Lane 1, Total soluble lysate of E. coli BL21(DE3)pLysS-pET-21d(+)-lukF-PV after induction with IPTG; Lane 2, Purified 6His-LukF-PV; Lane 3, Protein Mw standard. C. Purity of His-Tagged LukS-PV following Nickel Affinity chromatography. Lane 1, Protein Mw standard; Lane 2, Total soluble lysate of E. coli BL21(DE3)pLysS-pET-21d(+)-lukS-PV after induction with IPTG; Lane 3, water; Lanes 4, 5 & 6, Eluate without His-Tagged LukS-PV; Lanes 7, 8, 9 & 10, Eluate containing His-Tagged LukS-PV.

Figure 4

BioLogic LP chromatogram during Ni⁺⁺-affinity chromatographic purification of 6His-LukS-PV (upper) and 6His-LukF-PV (lower) in real-time. NOTE: AU = Absorbance unit, mS/cm = conductance of mobile phase. The inscriptions within the blue circles are the operational mode, i.e., S = start, H = hold, P = Pause, C = continue, and E = end. 6His-LukS-PV purification: The sign-post shows a low concentration of protein (0.8922 AU) in the eluate. B 6His-LukF-PV purification: The sign-post shows a high concentration of protein (5.000 AU) in the eluate.

Figure 5

Ion Exchange Chromatography (IEC) of 6His-LukS-PV. A AKTA Explorer chromatogram during ion exchange purification of 6His-LukS-PV. NOTE: The blue curve is the entire chromatographic plot itself. On the vertical axis, mS/cm is the conductivity or absorbance, while the green signal is the pressure. On the horizontal axis, the numbers in red (upper) are the identification of the tubes containing the protein, while the grey numbers (lower) is the timer (minutes). B Purity of 6His-LukS-PV following purification by IEC. Lanes 1, 2, 3 & 4, All of the eluate had bands of impurity between the 33 kDa and 25 kDa Mw marks. Lane M, Protein Mw standard.

Figure 6

Size Exclusion Chromatography (SEC) of 6His-LukS-PV. A AKTA Explorer Chromatographic plot during SEC purification of 6His-LukS-PV. NOTE: Vertical axis = absorbance, Horrizontal axis = Time (minutes). B Purity of 6His-LukS-PV following purification by SEC. Lane 1, 6His-LukS-PV completely pure without bands of impurity; Lane 2, water; Lane 3, Total soluble lysate of E. coli BL21(DE3)pLysS-pET-21d(+)-lukS-PV after induction with IPTG; Lane 4, E. coli BL21(DE3)pLysS-pET-21d(+)-lukS-PV before induction with IPTG, Lane M, Protein Mw standard.

Figure 7

Structures of 6His-LukSPV and 6His-LukF-PV as predicted by Protein Structure Prediction Server (PS)2 ( http://ps2.life.nctu.edu.tw/ ).