Open AccessCloning and expression of <i>Staphylococcus simulan</i>s lysostaphin enzyme gene in <i>Bacillus subtilis</i> WB600
Author(s) -
Babak Elyasi Far,
Mehran Ragheb,
Reza Rahbar,
Ladan Mafakher,
Neda Yousefi Nojookambari,
Spyridon Achinas,
Sajjad Yazdansetad
Publication year - 2021
Publication title -
aims microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.565
H-Index - 6
ISSN - 2471-1888
DOI - 10.3934/microbiol.2021017
Subject(s) - lysostaphin , bacillus subtilis , microbiology and biotechnology , plasmid , biology , recombinant dna , electroporation , expression vector , autolysin , gene , staphylococcus aureus , peptidoglycan , biochemistry , bacteria , genetics
Lysostaphin is a glycylglycine endopeptidase, secreted by Staphylococcus simulans , capable of specifically hydrolyzing pentaglycine crosslinks present in the peptidoglycan of the Staphylococcus aureus cell wall. In this paper, we describe the cloning and expression of the lysostaphin enzyme gene in Bacillus subtilis WB600 host using pWB980 expression system. Plasmid pACK1 of S. simulans was extracted using the alkaline lysis method. Lysostaphin gene was isolated by PCR and cloned into pTZ57R/T-Vector, then transformed into Escherichia coli DH5α. The amplified gene fragment and uncloned pWB980 vector were digested using Pst I and Xba 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 B. subtilis 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.