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Expression, Purification and Characterization of a Novel Hybrid Peptide CLP with Excellent Antibacterial Activity

CLP is a novel hybrid peptide derived from CM4, LL37 and TP5, with significantly reduced hemolytic activity and increased antibacterial activity than parental antimicrobial peptides. To avoid host toxicity and obtain high-level bio-production of CLP, we established a His-tagged SUMO fusion expressio...

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Autores principales: Cheng, Junhao, Ahmat, Marhaba, Guo, Henan, Wei, Xubiao, Zhang, Lulu, Cheng, Qiang, Zhang, Jing, Wang, Junyong, Si, Dayong, Zhang, Yueping, Zhang, Rijun
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8659006/
https://www.ncbi.nlm.nih.gov/pubmed/34885732
http://dx.doi.org/10.3390/molecules26237142
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author Cheng, Junhao
Ahmat, Marhaba
Guo, Henan
Wei, Xubiao
Zhang, Lulu
Cheng, Qiang
Zhang, Jing
Wang, Junyong
Si, Dayong
Zhang, Yueping
Zhang, Rijun
author_facet Cheng, Junhao
Ahmat, Marhaba
Guo, Henan
Wei, Xubiao
Zhang, Lulu
Cheng, Qiang
Zhang, Jing
Wang, Junyong
Si, Dayong
Zhang, Yueping
Zhang, Rijun
author_sort Cheng, Junhao
collection PubMed
description CLP is a novel hybrid peptide derived from CM4, LL37 and TP5, with significantly reduced hemolytic activity and increased antibacterial activity than parental antimicrobial peptides. To avoid host toxicity and obtain high-level bio-production of CLP, we established a His-tagged SUMO fusion expression system in Escherichia coli. The fusion protein can be purified using a Nickel column, cleaved by TEV protease, and further purified in flow-through of the Nickel column. As a result, the recombinant CLP with a yield of 27.56 mg/L and a purity of 93.6% was obtained. The purified CLP exhibits potent antimicrobial activity against gram+ and gram- bacteria. Furthermore, the result of propidium iodide staining and scanning electron microscopy (SEM) showed that CLP can induce the membrane permeabilization and cell death of Enterotoxigenic Escherichia coli (ETEC) K88. The analysis of thermal stability results showed that the antibacterial activity of CLP decreases slightly below 70 °C for 30 min. However, when the temperature was above 70 °C, the antibacterial activity was significantly decreased. In addition, the antibacterial activity of CLP was stable in the pH range from 4.0 to 9.0; however, when pH was below 4.0 and over 9.0, the activity of CLP decreased significantly. In the presence of various proteases, such as pepsin, papain, trypsin and proteinase K, the antibacterial activity of CLP remained above 46.2%. In summary, this study not only provides an effective strategy for high-level production of antimicrobial peptides and evaluates the interference factors that affect the biological activity of hybrid peptide CLP, but also paves the way for further exploration of the treatment of multidrug-resistant bacterial infections.
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spelling pubmed-86590062021-12-10 Expression, Purification and Characterization of a Novel Hybrid Peptide CLP with Excellent Antibacterial Activity Cheng, Junhao Ahmat, Marhaba Guo, Henan Wei, Xubiao Zhang, Lulu Cheng, Qiang Zhang, Jing Wang, Junyong Si, Dayong Zhang, Yueping Zhang, Rijun Molecules Article CLP is a novel hybrid peptide derived from CM4, LL37 and TP5, with significantly reduced hemolytic activity and increased antibacterial activity than parental antimicrobial peptides. To avoid host toxicity and obtain high-level bio-production of CLP, we established a His-tagged SUMO fusion expression system in Escherichia coli. The fusion protein can be purified using a Nickel column, cleaved by TEV protease, and further purified in flow-through of the Nickel column. As a result, the recombinant CLP with a yield of 27.56 mg/L and a purity of 93.6% was obtained. The purified CLP exhibits potent antimicrobial activity against gram+ and gram- bacteria. Furthermore, the result of propidium iodide staining and scanning electron microscopy (SEM) showed that CLP can induce the membrane permeabilization and cell death of Enterotoxigenic Escherichia coli (ETEC) K88. The analysis of thermal stability results showed that the antibacterial activity of CLP decreases slightly below 70 °C for 30 min. However, when the temperature was above 70 °C, the antibacterial activity was significantly decreased. In addition, the antibacterial activity of CLP was stable in the pH range from 4.0 to 9.0; however, when pH was below 4.0 and over 9.0, the activity of CLP decreased significantly. In the presence of various proteases, such as pepsin, papain, trypsin and proteinase K, the antibacterial activity of CLP remained above 46.2%. In summary, this study not only provides an effective strategy for high-level production of antimicrobial peptides and evaluates the interference factors that affect the biological activity of hybrid peptide CLP, but also paves the way for further exploration of the treatment of multidrug-resistant bacterial infections. MDPI 2021-11-25 /pmc/articles/PMC8659006/ /pubmed/34885732 http://dx.doi.org/10.3390/molecules26237142 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
Cheng, Junhao
Ahmat, Marhaba
Guo, Henan
Wei, Xubiao
Zhang, Lulu
Cheng, Qiang
Zhang, Jing
Wang, Junyong
Si, Dayong
Zhang, Yueping
Zhang, Rijun
Expression, Purification and Characterization of a Novel Hybrid Peptide CLP with Excellent Antibacterial Activity
title Expression, Purification and Characterization of a Novel Hybrid Peptide CLP with Excellent Antibacterial Activity
title_full Expression, Purification and Characterization of a Novel Hybrid Peptide CLP with Excellent Antibacterial Activity
title_fullStr Expression, Purification and Characterization of a Novel Hybrid Peptide CLP with Excellent Antibacterial Activity
title_full_unstemmed Expression, Purification and Characterization of a Novel Hybrid Peptide CLP with Excellent Antibacterial Activity
title_short Expression, Purification and Characterization of a Novel Hybrid Peptide CLP with Excellent Antibacterial Activity
title_sort expression, purification and characterization of a novel hybrid peptide clp with excellent antibacterial activity
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8659006/
https://www.ncbi.nlm.nih.gov/pubmed/34885732
http://dx.doi.org/10.3390/molecules26237142
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