Cargando…
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...
Autores principales: | , , , , , , , , , , |
---|---|
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 |
_version_ | 1784612863272288256 |
---|---|
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. |
format | Online Article Text |
id | pubmed-8659006 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT chengjunhao expressionpurificationandcharacterizationofanovelhybridpeptideclpwithexcellentantibacterialactivity AT ahmatmarhaba expressionpurificationandcharacterizationofanovelhybridpeptideclpwithexcellentantibacterialactivity AT guohenan expressionpurificationandcharacterizationofanovelhybridpeptideclpwithexcellentantibacterialactivity AT weixubiao expressionpurificationandcharacterizationofanovelhybridpeptideclpwithexcellentantibacterialactivity AT zhanglulu expressionpurificationandcharacterizationofanovelhybridpeptideclpwithexcellentantibacterialactivity AT chengqiang expressionpurificationandcharacterizationofanovelhybridpeptideclpwithexcellentantibacterialactivity AT zhangjing expressionpurificationandcharacterizationofanovelhybridpeptideclpwithexcellentantibacterialactivity AT wangjunyong expressionpurificationandcharacterizationofanovelhybridpeptideclpwithexcellentantibacterialactivity AT sidayong expressionpurificationandcharacterizationofanovelhybridpeptideclpwithexcellentantibacterialactivity AT zhangyueping expressionpurificationandcharacterizationofanovelhybridpeptideclpwithexcellentantibacterialactivity AT zhangrijun expressionpurificationandcharacterizationofanovelhybridpeptideclpwithexcellentantibacterialactivity |