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Production and Purification of Two Bioactive Antimicrobial Peptides Using a Two-Step Approach Involving an Elastin-Like Fusion Tag

Antimicrobial resistance is an increasing global threat, demanding new therapeutic biomolecules against multidrug-resistant bacteria. Antimicrobial peptides (AMPs) are promising candidates for a new generation of antibiotics, but their potential application is still in its infancy, mostly due to lim...

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Autores principales: Pereira, Ana Margarida, da Costa, André, Dias, Simoni Campos, Casal, Margarida, Machado, Raul
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8541314/
https://www.ncbi.nlm.nih.gov/pubmed/34681180
http://dx.doi.org/10.3390/ph14100956
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author Pereira, Ana Margarida
da Costa, André
Dias, Simoni Campos
Casal, Margarida
Machado, Raul
author_facet Pereira, Ana Margarida
da Costa, André
Dias, Simoni Campos
Casal, Margarida
Machado, Raul
author_sort Pereira, Ana Margarida
collection PubMed
description Antimicrobial resistance is an increasing global threat, demanding new therapeutic biomolecules against multidrug-resistant bacteria. Antimicrobial peptides (AMPs) are promising candidates for a new generation of antibiotics, but their potential application is still in its infancy, mostly due to limitations associated with large-scale production. The use of recombinant DNA technology for the production of AMPs fused with polymer tags presents the advantage of high-yield production and cost-efficient purification processes at high recovery rates. Owing to their unique properties, we explored the use of an elastin-like recombinamer (ELR) as a fusion partner for the production and isolation of two different AMPs (ABP-CM4 and Synoeca-MP), with an interspacing formic acid cleavage site. Recombinant AMP-ELR proteins were overproduced in Escherichia coli and efficiently purified by temperature cycles. The introduction of a formic acid cleavage site allowed the isolation of AMPs, resorting to a two-step methodology involving temperature cycles and a simple size-exclusion purification step. This simple and easy-to-implement purification method was demonstrated to result in high recovery rates of bioactive AMPs. The minimum inhibitory concentration (MIC) of the free AMPs was determined against seven different bacteria of clinical relevance (Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and two Burkholderia cenocepacia strains), in accordance with the EUCAST/CLSI antimicrobial susceptibility testing standards. All the bacterial strains (except for Pseudomonas aeruginosa) were demonstrated to be susceptible to ABP-CM4, including a resistant Burkholderia cenocepacia clinical strain. As for Synoeca-MP, although it did not inhibit the growth of Pseudomonas aeruginosa or Klebsiella pneumoniae, it was demonstrated to be highly active against the remaining bacteria. The present work provides the basis for the development of an efficient and up-scalable biotechnological platform for the production and purification of active AMPs against clinically relevant bacteria.
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spelling pubmed-85413142021-10-24 Production and Purification of Two Bioactive Antimicrobial Peptides Using a Two-Step Approach Involving an Elastin-Like Fusion Tag Pereira, Ana Margarida da Costa, André Dias, Simoni Campos Casal, Margarida Machado, Raul Pharmaceuticals (Basel) Article Antimicrobial resistance is an increasing global threat, demanding new therapeutic biomolecules against multidrug-resistant bacteria. Antimicrobial peptides (AMPs) are promising candidates for a new generation of antibiotics, but their potential application is still in its infancy, mostly due to limitations associated with large-scale production. The use of recombinant DNA technology for the production of AMPs fused with polymer tags presents the advantage of high-yield production and cost-efficient purification processes at high recovery rates. Owing to their unique properties, we explored the use of an elastin-like recombinamer (ELR) as a fusion partner for the production and isolation of two different AMPs (ABP-CM4 and Synoeca-MP), with an interspacing formic acid cleavage site. Recombinant AMP-ELR proteins were overproduced in Escherichia coli and efficiently purified by temperature cycles. The introduction of a formic acid cleavage site allowed the isolation of AMPs, resorting to a two-step methodology involving temperature cycles and a simple size-exclusion purification step. This simple and easy-to-implement purification method was demonstrated to result in high recovery rates of bioactive AMPs. The minimum inhibitory concentration (MIC) of the free AMPs was determined against seven different bacteria of clinical relevance (Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and two Burkholderia cenocepacia strains), in accordance with the EUCAST/CLSI antimicrobial susceptibility testing standards. All the bacterial strains (except for Pseudomonas aeruginosa) were demonstrated to be susceptible to ABP-CM4, including a resistant Burkholderia cenocepacia clinical strain. As for Synoeca-MP, although it did not inhibit the growth of Pseudomonas aeruginosa or Klebsiella pneumoniae, it was demonstrated to be highly active against the remaining bacteria. The present work provides the basis for the development of an efficient and up-scalable biotechnological platform for the production and purification of active AMPs against clinically relevant bacteria. MDPI 2021-09-23 /pmc/articles/PMC8541314/ /pubmed/34681180 http://dx.doi.org/10.3390/ph14100956 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Pereira, Ana Margarida
da Costa, André
Dias, Simoni Campos
Casal, Margarida
Machado, Raul
Production and Purification of Two Bioactive Antimicrobial Peptides Using a Two-Step Approach Involving an Elastin-Like Fusion Tag
title Production and Purification of Two Bioactive Antimicrobial Peptides Using a Two-Step Approach Involving an Elastin-Like Fusion Tag
title_full Production and Purification of Two Bioactive Antimicrobial Peptides Using a Two-Step Approach Involving an Elastin-Like Fusion Tag
title_fullStr Production and Purification of Two Bioactive Antimicrobial Peptides Using a Two-Step Approach Involving an Elastin-Like Fusion Tag
title_full_unstemmed Production and Purification of Two Bioactive Antimicrobial Peptides Using a Two-Step Approach Involving an Elastin-Like Fusion Tag
title_short Production and Purification of Two Bioactive Antimicrobial Peptides Using a Two-Step Approach Involving an Elastin-Like Fusion Tag
title_sort production and purification of two bioactive antimicrobial peptides using a two-step approach involving an elastin-like fusion tag
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8541314/
https://www.ncbi.nlm.nih.gov/pubmed/34681180
http://dx.doi.org/10.3390/ph14100956
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