2.1. Computer Modeling
The structure of SpA protein according to the amino acid sequence in this study that comprises five repeat domains of 295 amino acid residues in length (SpA295) was predicted by ModWeb server (Pieper at al., 2014). The nucleotide sequence of the Lpp'-ompA-Spa construct was submitted to genebank with the accession number: MT680197. The Geometric coordinates of X-ray crystallography of IgG were obtained from RCSB protein data bank with the access code: 4ZNC.
2.2. Computational condition of docking and molecular dynamic simulation
To provide the stable structure of Lpp’-OmpA-SPA295, this complex was subjected to molecular dynamic (MD) simulation for 30 ns. MD simulation was performed by GROMACS 5.0.5 software (van Der Spoel et al., 2005) and OPLSAA force field similar to that shown in the previous study (Ghahremanifard et al., 2018; Hashemzadeh et al., 2018; Fasehee et al., 2018) The molecules were placed in a dodecahedron box containing the water molecule in TIP3P model. In order to create the ionic conditions of 0.15 molar, water molecules were replaced with Na+ and Cl- ions and the total charge of system was neutralized. The initial energy minimization was performed using the steepest descent algorithm. After that, the NVT simulation was performed for 50 ps and followed by the NPT ensemble for 30 ns. In order to maintain the temperature of 300° K and the pressure of 1 bar, nose-hover thermostat and Berendsen barostat were used, respectively. R =1.2 was considered for electrostatic and van der Waals interactions.
The stable structure of Lpp'-OmpA-SPA295 from primary MD simulation was used to investifate the interaction of this structure with IgG. For this purpose, the HDOCK server (Yan et al., 2014) was employed to investigate Lpp'-OmpA-SpA295-IgG interaction according to default parameters of protein–protein free docking hybrid algorithm of template-based modeling.
The complex obtained from the HDOCK server was subjected to 30 ns molecular dynamic simulation under the conditions used for the primary MD simulation.
All structures visualized by the Discovery studio. The number of hydrogen bond (H-bond) formed between acceptor and donor atoms is measured using the geometrical criteria of a donor-acceptor distance less than 3.5 Å by RING 2.0 web server (The RING 2.0 web server for high quality residue interaction networks).
2.3. Materials used in Experimental Model
List of the primer pairs, bacterial strains and plasmids used in this study are listedin Table 1. The SpA gene were amplified from the genomic DNA of S. aureus (ATCC 6538) as a template, the Pfu DNA polymerase (Fermentas, Germany) and primers shown in Table 1. For design of growth curves and optimization tests, bacterial cultures were grown in Luria-Bertani (LB) medium containing 50 mg/ml kanamycin sulfate. Isopropyl ß--D-thiogalactopyranoside (IPTG) was used to induce the expression of recombinant protein n. In this study Human serum was used for binding analysis.
2.4. Construction of plasmids, and protein expression
The Lpp′-OmpA fragment was amplified by PCR using LPOA1 and LPOTA primers to construct pLOAa plasmid, previously made from the pET-LOA plasmid, containing a combining of the first nine N- terminal amino acids of Lpp and amino acids 46 to 159 of OmpA were used as a template. The 381 bp PCR product was digested with NdeI-EcoRI restriction enzymes, followed by ligation into the previously digested pET26b vector.
PCR was carried out as per following conditions: Initial denature for 5 min at 94C, amplify 30 cyclesof 45 s at 94 °C, annealing foe 45 s at 68 °C, extension for 60s at 72 °C, and final extension for10 min at 72 °C on an MWG AG, Biotech, Primus 96 system (Germany). For gene cloning, the truncated SpA gene was amplified using primers PAF, PAR from ~ 890 bp, S. aureus genome as a template (100 ng/25 µl), and purified. To prepare the final construct of plasmid, SpA fragment (890 bp) and pLOAa were digested by EcoRI and XhoI and followed by ligation into the plasmid pLOAa. The vector was labeled as pLOA-PA pLOA-PA. Transformation of the vector into E. coli was carried out. It used CaCl2-mediated procedure. Overnight cultures of recombinant bacterial inoculated into 1 liter of fresh LB medium which contains 50 μg/ml kanamycin sulfate. To express fusion proteins, cultures were induced by using IPTG 0.1 mM, for 16 h. Centrifugation was used at 10000g for 2 minute to harvest the cells.
2.5. Preparation of surface-engineered E. coli displaying SpA protein
A single colony of the recombinant E. coli harboring plasmids pLOA-PA was grown overnight in the LB media containing 35 mg/ml of kanamycin and was then inoculated into the fresh medium and continued for 8hr further incubation.
The bacterial culture was collected by centrifugation at 5000xg for 10 min followed by a washing step with 10 ml PBS buffer by resuspended in PBS following a washing step. This was repeated for three more times.
The bacteria pellet was resuspended in 10 ml of PBS + 0.02% sodium azide, transferred to a 250 mL erlenmeyer flask and stirred at room temperature. Formaldehyde solution was added to give 1.5% final concentration and stirring was continued for 80 minutes at room temperature. Formaldehyde was removed by washing the suspension with 15 ml of 1X PBS. After discarding the supernatant, the pellet was washed in PBS + 0.02% sodium azide as a 10% (w/v) followed by a 5 min centrifugation at 5000Xg. After resuspension of the bacterial pellet in a 100mM Tris-HCl buffer pH 8 containing 10% glycerol, it was stored at 4 ºC.
2.6. IgG-binding assay
The affinity and IgG-binding ability of the protein A-displaying E. coli was examined using IgG-binding assay with rabbit sera. The surface-engineered E. coli was washed with 1mL of suspension buffer (100mM Tris-HCl pH 8). The pH of rabbit sera was increased to 7.5–8 by 1M Tris of pH 8 and it then added to the bacterial suspension followed by incubation for one hour at 4°C. The surface-engineered bacteria were washed with 100 mM Tris-HCl of pH 8. Elution buffer (100mM glycine of pH 3 containing 1M KCl) was used to release the bound IgGs from surface of the bacterial. Eluted fraction was dialyzed in 10 mM Tris-HCl and then resolved on SDS-PAGE prepared according to Laemmli (1970) and then stained with Coomassie Brilliant Blue R-250. E. coli transformed with the parental plasmid pLpp’-OmpA without IgG-binding domain of SpA was used as a negative control.