Cloning and expression of <i>Staphylococcus simulan</i>s lysostaphin enzyme gene in <i>Bacillus subtilis</i> WB600

Babak Elyasi Far; Mehran Ragheb; Reza Rahbar; Ladan Mafakher; Neda Yousefi Nojookambari; Spyridon Achinas; Sajjad Yazdansetad; Babak Elyasi Far; Mehran Ragheb; Reza Rahbar; Ladan Mafakher; Neda Yousefi Nojookambari; Spyridon Achinas; Sajjad Yazdansetad

doi:10.3934/microbiol.2021017

AIMS Microbiology

2021, Volume 7, Issue 3: 271-283. doi: 10.3934/microbiol.2021017

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Research article Special Issues

Cloning and expression of Staphylococcus simulans lysostaphin enzyme gene in Bacillus subtilis WB600

1.
Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran
2.
Student Research Committee, Golestan University of Medical Sciences, Gorgan, Iran
3.
Medical Plant Research Center, Ahvaz Jundishapur University of Medical Science, Ahvaz, Iran
4.
Department of Microbiology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
5.
Faculty of Science and Engineering, University of Groningen, Groningen, The Netherlands
6.
Laboratory Sciences Research Center, Golestan University of Medical Sciences, Gorgan, Iran

Received: 01 May 2021 Accepted: 16 July 2021 Published: 23 July 2021

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.
- Bacillus subtilis,
- Lysostaphin,
- Staphylococcus aureus,
- plasmids,
- enzyme stability
Citation: Babak Elyasi Far, Mehran Ragheb, Reza Rahbar, Ladan Mafakher, Neda Yousefi Nojookambari, Spyridon Achinas, Sajjad Yazdansetad. Cloning and expression of Staphylococcus simulans lysostaphin enzyme gene in Bacillus subtilis WB600[J]. AIMS Microbiology, 2021, 7(3): 271-283. doi: 10.3934/microbiol.2021017

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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.

Acknowledgments

We hereby appreciate to Vice-chancellor for Research & Technology of Golestan University of Medical Sciences (GOUMS) for valuable contributions in this research. A very special thanks goes to the Faculty of Science and Engineering, University of Groningen, The Netherlands for the meritorious collaboration.

Conflicts of interest

The authors declare that there is no conflict of interest regarding the publication of this article.

Authors' contributions

Babak Elyasi Far, Mehran Ragheb, Reza Rahbar, Ladan Mafakher, and Neda Yousefi Nojookambari performed experiments. All authors contributed to the acquisition of data, analysis, and interpretation. Babak Elyasi Far drafted and provided critical revision of the article. Sajjad Yazdansetad wrote the paper and contributed to the conception and design of the study. Spyridon Achinas provided the final approval of the version to publish.

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