<abstract xmlns="http://www.w3.org/1999/xhtml"><p>Antimicrobial peptides are natural substances that have played a role in the development of the adaptive immune system, and are currently involved in the prevention of infections, through their direct antimicrobial and immunomodulatory properties. While the amino acid composition and spatial structure vary, most antibacterial peptides have a positive surface charge, which allows them to bind to the negative bacterial membranes. Buforin II is a widely studied antimicrobial peptide first obtained through the structural modification of buforin I, a peptide isolated from <italic>Bufo gargarizans</italic>. The peptide showed significant antibacterial activity against Gram-positive and Gram-negative bacterial strains. The mechanism of action of buforin II differs from that of other antimicrobial peptides, as it binds directly to bacterial DNA and RNA. The aim of our study was to obtain recombinant buforin II with a ubiquitin fusion partner, through heterologous expression in <italic>Escherichia coli</italic> Rosetta™ (DE3)pLysS cells, using a laboratory scale bioreactor. The incubation of expression host cells in a bioreactor allowed the constant monitoring and control of the process parameters, leading to high biomass levels and an increased production rate of the peptide. The parameters used during incubation were: 37°C, pH=6.9 and dissolved oxygen level above 40%. Purification of the recombinant protein was accomplished by affinity chromatography using a Ni-chelate solid phase to which the 10xHistag of our construct showed affinity. Method optimisation consisted in the use of gradient and linear elution, of which the latter was found to be more effective. Digestion of the fusion partner from the target peptide was performed with ubiquitin carboxyl-terminal hydrolase enzyme. The expression and purification protocols developed in our experiment allow the production of a significant amount of buforin II, allowing its use for further research. Furthermore, the presented methods could be suitable for industrial production of the recombinant peptide..</p></abstract>

Antimicrobial peptides are natural substances that have played a role in the development of the adaptive immune system, and are currently involved in the prevention of infections, through their direct antimicrobial and immunomodulatory properties. While the amino acid composition and spatial structure vary, most antibacterial peptides have a positive surface charge, which allows them to bind to the negative bacterial membranes. Buforin II is a widely studied antimicrobial peptide first obtained through the structural modification of buforin I, a peptide isolated from Bufo gargarizans. The peptide showed significant antibacterial activity against Gram-positive and Gram-negative bacterial strains. The mechanism of action of buforin II differs from that of other antimicrobial peptides, as it binds directly to bacterial DNA and RNA. The aim of our study was to obtain recombinant buforin II with a ubiquitin fusion partner, through heterologous expression in Escherichia coli Rosetta™ (DE3)pLysS cells, using a laboratory scale bioreactor. The incubation of expression host cells in a bioreactor allowed the constant monitoring and control of the process parameters, leading to high biomass levels and an increased production rate of the peptide. The parameters used during incubation were: 37°C, pH=6.9 and dissolved oxygen level above 40%. Purification of the recombinant protein was accomplished by affinity chromatography using a Ni-chelate solid phase to which the 10xHistag of our construct showed affinity. Method optimisation consisted in the use of gradient and linear elution, of which the latter was found to be more effective. Digestion of the fusion partner from the target peptide was performed with ubiquitin carboxyl-terminal hydrolase enzyme. The expression and purification protocols developed in our experiment allow the production of a significant amount of buforin II, allowing its use for further research. Furthermore, the presented methods could be suitable for industrial production of the recombinant peptide..

Heterologous Expression and Purification of the Antimicrobial Peptide Buforin II

University of Medicine, Pharmacy, Science and Technology of Târgu Mureș

Department of General and Inorganic Chemistry

Department of Pharmaceutical Industry and Management

Sapientia Hungarian University of Transylvania

Faculty of Economics, Socio-Human Sciences and Engineering, Miercurea Ciuc, Genetic Engineering specialty

Faculty of Economics, Socio-Human Sciences and Engineering, Miercurea Ciuc, Department of Bioengineering

University of Medicine, Pharmacy, Sciences and Technology of Târgu Mureș

Bulletin of Medical Sciences

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

{"article-title":"Heterologous Expression and Purification of the Antimicrobial Peptide Buforin II"}

Antimicrobial peptides are natural substances that have played a role in the development of the adaptive immune system, and are currently involved in the...

Heterologous Expression and Purification of the Antimicrobial Peptide Buforin II

Publié en ligne: 03 déc. 2020

Pages: 119 - 125

DOI: https://doi.org/10.2478/orvtudert-2019-0010

Mots clés
buforin II, heterologous expression, antimicrobial peptide, AMP

© 2019 Boda Ferenc-András et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Heterologous Expression and Purification of the Antimicrobial Peptide Buforin II

Publié en ligne: 03 déc. 2020

Pages: 119 - 125

DOI: https://doi.org/10.2478/orvtudert-2019-0010

Mots clésbuforin II, heterologous expression, antimicrobial peptide, AMP

© 2019 Boda Ferenc-András et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Mots clés
buforin II, heterologous expression, antimicrobial peptide, AMP