Cloning and expression of <i>Staphylococcus simulan</i>s lysostaphin enzyme gene in <i>Bacillus subtilis</i> WB600
2021
Babak Elyasi Far | Mehran Ragheb | Reza Rahbar | Ladan Mafakher | Neda Yousefi Nojookambari | Spyridon Achinas | Sajjad Yazdansetad
Lysostaphin is a glycylglycine endopeptidase, secreted by <i>Staphylococcus simulans</i>, capable of specifically hydrolyzing pentaglycine crosslinks present in the peptidoglycan of the <i>Staphylococcus aureus</i> cell wall. In this paper, we describe the cloning and expression of the lysostaphin enzyme gene in <i>Bacillus subtilis</i> WB600 host using pWB980 expression system. Plasmid pACK1 of <i>S. simulans</i> was extracted using the alkaline lysis method. Lysostaphin gene was isolated by PCR and cloned into pTZ57R/T-Vector, then transformed into <i>Escherichia coli</i> DH5α. The amplified gene fragment and uncloned pWB980 vector were digested using <i>Pst</i>I and <i>Xba</i>І enzymes and purified. The restricted gene fragment was ligated into the pWB980 expression vector by the standard protocols, then the recombinant plasmid was transformed into <i>B. subtilis</i> WB600 using electroporation method. The recombinant protein was evaluated by the SDS-PAGE method and confirmed by western immunoblot. Analysis of the target protein showed a band corresponding to 27-kDa r-lysostaphin. Protein content was estimated 91 mg/L by Bradford assay. The recombinant lysostaphin represented 90% of its maximum activity at 40 °C and displayed good thermostability by keeping about 80% of its maximum activity at 45 °C. Heat residual activity assay of recombinant lysostaphin demonstrated that the enzyme stability was up to 40 °C and showed good stability at 40 °C for 16 h incubation.
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