Functional expression and purification of tailor-made chimeric endolysin with the broad antibacterial spectrum
- PDF / 2,837,866 Bytes
- 13 Pages / 595.276 x 790.866 pts Page_size
- 3 Downloads / 164 Views
ORIGINAL ARTICLE
Functional expression and purification of tailor-made chimeric endolysin with the broad antibacterial spectrum Michaela Mancoš 1,2
&
Zuzana Šramková 1,3 & Darina Peterková 1 & Barbora Vidová 4 & Andrej Godány 1
Received: 17 December 2019 / Accepted: 30 April 2020 # Institute of Molecular Biology, Slovak Academy of Sciences 2020
Abstract Developing chimeric lysins with a wide lytic spectrum is important in fighting against Gram-negative pathogenic bacteria. In the present work, a novel chimerical lysin, LytAmfi, was constructed by fusing the bacteriophage endolysin Lyt μ1/6 with an amphipathic cationic peptide derived from T4 lysozyme. The result showed that the LytAmfi had not only lytic activity similar to parental Streptomyces aureofaciens endolysin Lyt μ1/6 but also broadened lytic activity against Gram-negative strains. Our work demonstrated that generating a novel chimeric lysin with an extended lytic spectrum was workable by fusing an active modular endolysin with an amphipathic cationic peptide that could penetrate the outer membrane of Gram-negative bacteria including Escherichia coli, Pseudomonas agglomerans, Acinetobacter lwoffii, Hafnia alvei, Citrobacter freundii and enable access of the lysin to lyse the bacteria ‘from within’ the host cell without pre-treatment of its outer membrane. Therefore, lysins with an extended spectrum of lytic activity would be of appreciable therapeutic value. Keywords Actinophage μ1/6 . T4 lysozyme . Cationic tag . In silico analysis . Chimeric lysin . Heterologous protein expression
Abbreviations CBD CD-search CFU ECD FPLC GFP HEPES IPTG LB LBS
NCBI C-terminal cell-wall binding domain Conserved domain Search service Colony-forming unit SN-terminal catalytic domain Fast protein liquid chromatography Green fluorescent protein 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane sulfonic acid Isopropyl β-D-1-thiogalactopyranoside Lysogeny broth Lysogeny broth with high salt
Ni-NTA OD PCR PDB PGRP PHAGE PSI-BLAST SDS-PAGE TB TBG
National Center for Biotechnology Information Nickel-nitrilotriacetic acid Optical density Polymerase chain reaction Protein data bank Peptidoglycan recognition proteins Bacteriophage Position-specific iterated basic local alignment search tool Sodium dodecyl sulphate polyacrylamide gel electrophoresis Terrific broth Terrific broth with glycylglycine
* Michaela Mancoš [email protected]
Introduction 1
Department of Biology, University of SS. Cyril and Methodius, J. Herdu 2, Trnava SK-91701, Slovak Republic
2
Regional Authority of Public Health in Banská Bystrica, Cesta k nemocnici 1, BanskáBystrica SK-7401, Slovak Republic
3
Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, Bratislava SK-8455, Slovak Republic
4
Barbora Vidová – AWIT, Budyšínska 3062/20, Bratislava SK-83103, Slovak Republic
Bacteriophage (phage) endolysins, specific peptidoglycan hydrolases, have the potential to become powerful enzybiotics against multi-drug resistant bacteria. They are produced by phages at the end of t
Data Loading...
