2.1. Bacterial strains, plasmids, enzymes, and reagents
Staphylococcus simulans biovar staphylolyticus (ATCC 27848) as the source of the lysostaphin gene was obtained from Persian Type Culture Collection (PTCC, Tehran, Iran). E. coli DH5α was purchased from Novagen Company (Novagen, Madison, WI, USA). Bacillus subtilis strain WB600, the bioengineered strain with the deficient in six extracellular proteases(15), was kindly provided by Professor Sui-Lam Wong (University of Calgary, Canada). The pTZ57R/T vector (Fig. 1) provided by Fermentas Co., Ltd (Fermentas, Vilnius, Lithuania). The expression vector pWB980 (Fig. 2) was prepared from Nova Lifetech Inc., Hong Kong. The restriction enzymes PstI, XbaІ and EcoRІ were purchased from Vivantis (Vivantis, Malaysia). T4 DNA Ligase, pfuTurbo DNA polymerase, and Taq DNA polymerase enzymes were bought from GeneON (GeneON, Nurnberg, Germany), Fermentas (Fermentas, Vilnius, Lithuania), and Cinnagen (Cinnagen, Tehran, Iran), respectively. Oligonucleotide primers were synthesized by TAG Copenhagen, Denmark. DNA Ladder O'GeneRuler 1 kb Plus was purchased from Thermo Scientific™. Broad range unstained protein standard marker was provided by Fermentas Co., Ltd. All culture media were provided by HiMedia Laboratories Pvt. Ltd., India.
2.2. Plasmid extraction and PCR cloning of lysostaphin gene
The plasmid was extracted from S. simulans using an optimized alkaline lysis method. Briefly, the bacterial cell pellet was resuspended in 100 µL resuspension buffer (Tris-Acetat 40 mM, Sodium-EDTA 2 mM, pH 7.9), then the cells were lysed with 200 µL lysis solution (Tris 50 mM, SDS 3%, pH 12.6) and incubated in a water bath at 65℃ for 15 min, and then neutralized with 450 µL of cold potassium acetate 5 M, glacial acetic acid, pH 4.8. The mixture was centrifuged at 13,000 rpm for 10 min at 4 °C. The supernatant was mixed with an equal volume of isopropanol and placed at − 20 °C for 20 min. The mixture was centrifuged at 13,000 rpm for 10 min at 25 °C. The supernatant was removed and 200 µL of 70% ethanol was added to the pellet and centrifuged at 13,000 rpm for 5 min at 25 °C. The pellet was resuspended in 50 µL dH2O. Extracted plasmid was visualized on a 1% agarose gel stained with ethidium bromide and run at 90 V for 50 min.
Specific primers for PCR amplification of the lysostaphin gene were designed according to the known lysostaphin gene sequence from S. simulans deposited in GenBank under accession No. M15686.
The forward primer (5'-AGATCTAGAGCTGCAACACATGAACATTCAGCA-3'), XbaІ restriction site underlined in the primer sequence, and the reverse primer (5'-TCACTGCAGCTTTATAGTTCCCCAAAGAACAC-3'), PstI restriction site underlined in the primer sequence, were used for PCR amplification of the lysostaphin gene. The PCR reaction mixture was prepared in a total volume of 25 µl containing 10 nanograms (ng) of genomic DNA, 10 mM dNTP mix, 10 pM of each oligonucleotide primer, and 2.5 U Taq DNA polymerase in PCR buffer with 20 mM MgSO4. The PCR reaction was carried out in thermal cycler Peqlab Primus 25 (Peqlab Primus 25, UK) under the following steps: 5 min initial denaturation at 94 °C, followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 58 °C for 1 min, synthesis at 72 °C for 1 min, followed by 10 min elongation at 72 °C. PCR product was electrophoresed on 1% (w/v) ethidium bromide-stained agarose gel and purified using GeneJET PCR Purification Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions.
2.3. Cloning of lysostaphin gene
The pTZ57R/T vector and E. coli DH5α were used for the lysostaphin gene cloning, pWB980 expression vector and B. subtilis WB600 were utilized for the subcloning. The ligation reaction was done between the PCR product ⁓600 nanogram (ng) and pTZ57R/T vector ⁓200 ng. The ligated vector was transformed into E. coli DH5α by cold CaCl2 shock method. Then, 100 µl of the transformed E. coli DH5α was cultured aerobically in Luria–Bertani (LB) medium supplemented with 100 µg/ml ampicillin, 30 µg/ml X-gal, and 2 mM IPTG at 37 °C for overnight. The white colonies on the medium were designated and subcultured, followed by the recombinant plasmids were extracted and analyzed. The lysostaphin gene fragment was digested by PstI and XbaІ enzymes from T-vector. The expression vector pWB980 was also digested by the same enzymes (PstI and XbaІ) and purified by GeneJET Purification Kit, then ligated to the pWB980 expression vector by the standard protocols.
2.4. Transformation into Bacillus subtilis and construction of expression vector
B. subtilis WB600 was used for extracellular production of recombinant lysostaphin. The ligation reaction was carried out with 1 µg pWB980 plasmid and 3 µg gene fragment. Afterward, 20 µl of the ligation mixture was transformed into B. subtilis by electroporation method at 8 milliseconds and 950 V (Gene Pulser Xcell™ Electroporation System, Bio-Rad, USA). Finally, 200 µl of the transformed B. subtilis was aerobically cultured in LB medium supplemented with 10 µg/ml kanamycin (Sigma-Aldrich) at 37 °C for 16 h.
2.5. Expression of lysostaphin
B. subtilis cells with pWB980 plasmid encoding lysostaphin gene were grown in LB medium supplemented with 10 µg/ml kanamycin on a rotator shaker (180 rpm) at 37 °C to reach an optical density (OD) of 1.2–1.5 at a wavelength of 600 nm. The cells were centrifuged and the supernatant was fractionated by adding solid (NH4)2SO4 at 4℃. The protein was pelleted and resuspended in phosphate buffer solution 0.1 M, pH 7.2.
2.6. SDS-PAGE and Western blotting
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was achieved on 12% (w/v) resolving gel as described by Laemmli(16). The recombinant protein band was monitored by using SDS-PAGE following Coomassie brilliant blue G-250 staining method. The protein concentration was measured by Bradford assay using bovine serum albumin (BSA) as the standard(17). For the immunoblotting assay, a replicate gel was moved onto a nitrocellulose membrane (Macherey-Nagel™ Porablot NCP, Germany) for 1.5 h at 320 V, soaked in 5% BSA blocking solution for overnight at 4℃, then washed 3 times by TBS-T (Tris-HCl 20 mM, NaCl 150 mM, pH 7.5-Tween 0.05%). The blots were incubated with a dilution of 1:100 antiserum, followed by a 1:2000 dilution of HRP- labeled Goat Anti-Rabbit IgG (SouthernBiotech, USA) in TBS-T for 1 h. The signals were developed with DAB (3,3′-Diaminobenzidine) (Sigma-Aldrich, USA) and H2O2 substrate.
2.7. Bacteriolytic activity of r-lysostaphin
The bacteriolytic activity of r-lysostaphin was assayed by spectrophotometric measurements of turbidity as previously described by Marova and Kovar(18). Briefly, the reaction mixture, containing 6 ml suspension of S. aureus DSM 1104 diluted in PBS (Phosphate-buffered saline 0.1 M, pH 7.2) to reach OD620 nm=0.25, was preincubated at 37℃ for 10 min and then 20 ml of r-lysostaphin was added. The changes in turbidity of the reaction mixture were determined. One unit of lysostaphin activity was defined as a total preparation affecting 50% turbidity reduction of the bacterial cell suspension at absorbance of 620 nm (A620 nm) within 10 min at 37℃ in a 10 mm cuvette. The pH and temperature parameters on recombinant enzyme activity were assayed in the ranges of 5.0–10.0 and 20–60℃, respectively. The residual activity of r-lysostaphin was also studied at different temperatures and time treatments.