Abstract
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 PstI and XbaІ 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.
Highlights
Lysostaphin is a zinc-metalloprotease glycylglycine endopeptidase enzyme originally secreted by Staphylococcus simulans biovar staphylolyticus
Recombinant plasmid clones carrying the lysostaphin gene were double digested by XbaІ and PstI enzymes, the gene fragment was properly ligated into the pWB980 expression vector with the appropriate orientation under the control of P43 promoter
The pWB980 vector contains an auto-inducible P43 promoter, sacB signal sequence, multiple cloning site (MCS), and kanamycin-resistance marker derived from B. subtilis [19]
Summary
Lysostaphin is a zinc-metalloprotease glycylglycine endopeptidase enzyme originally secreted by Staphylococcus simulans biovar staphylolyticus. Lysostaphin is initially produced as a preproenzyme of 493 amino acids with three domains: an N-terminal domain as the secretion signal peptide of 36 amino acid residues, a proenzyme of 211 amino acid residues harboring 15 tandem repeats (TRs) of 13 amino acids, and a mature enzyme of 246 amino acid residues. The mature lysostaphin consists of two domains: an N-terminal peptidase domain involved in the catalytic activity of the enzyme, and a C-terminal domain binding to the peptidoglycan [1]. Previous studies reported that the C-terminal domain with 92 amino acids not involved in the enzymatic activity; it plays an important role in directing lysostaphin to the S. aureus cell wall [2]. We examine the cloning and expression of the S. simulans lysostaphin enzyme gene in AIMS Microbiology
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