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High-Yield Soluble Expression and Simple Purification of the Antimicrobial Peptide OG2 Using the Intein System in Escherichia coli

OG2 is a modified antimicrobial peptide, that is, derived from the frog peptide Palustrin-OG1. It has high antimicrobial activity and low cytotoxicity, and it is therefore promising as a therapeutic agent. Both prokaryotic (Escherichia coli) and eukaryotic (Pichia pastoris) production host systems w...

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Autores principales: Xie, Yong-Gang, Han, Fei-Fei, Luan, Chao, Zhang, Hai-Wen, Feng, Jie, Choi, Young-Jun, Groleau, Denis, Wang, Yi-Zhen
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Hindawi Publishing Corporation 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3713655/
https://www.ncbi.nlm.nih.gov/pubmed/23936842
http://dx.doi.org/10.1155/2013/754319
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author Xie, Yong-Gang
Han, Fei-Fei
Luan, Chao
Zhang, Hai-Wen
Feng, Jie
Choi, Young-Jun
Groleau, Denis
Wang, Yi-Zhen
author_facet Xie, Yong-Gang
Han, Fei-Fei
Luan, Chao
Zhang, Hai-Wen
Feng, Jie
Choi, Young-Jun
Groleau, Denis
Wang, Yi-Zhen
author_sort Xie, Yong-Gang
collection PubMed
description OG2 is a modified antimicrobial peptide, that is, derived from the frog peptide Palustrin-OG1. It has high antimicrobial activity and low cytotoxicity, and it is therefore promising as a therapeutic agent. Both prokaryotic (Escherichia coli) and eukaryotic (Pichia pastoris) production host systems were used to produce OG2 in our previous study; however, it was difficult to achieve high expression yields and efficient purification. In this study, we achieved high-yield OG2 expression using the intein fusion system. The optimized OG2 gene was cloned into the pTWIN1 vector to generate pTWIN-OG2-intein2 (C-terminal fusion vector) and pTWIN-intein1-OG2 (N-terminal fusion vector). Nearly 70% of the expressed OG2-intein2 was soluble after the IPTG concentration and induction temperature were decreased, whereas only 42% of the expressed of intein1-OG2 was soluble. Up to 75 mg of OG2-intein2 was obtained from a 1 l culture, and 85% of the protein was cleaved by 100 mM DTT. Intein1-OG2 was less amenable to cleavage due to the inhibition of cleavage by the N-terminal amino acid of OG2. The purified OG2 exhibited strong antimicrobial activity against E. coli K88. The intein system is the best currently available system for the cost-effective production of OG2.
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spelling pubmed-37136552013-08-09 High-Yield Soluble Expression and Simple Purification of the Antimicrobial Peptide OG2 Using the Intein System in Escherichia coli Xie, Yong-Gang Han, Fei-Fei Luan, Chao Zhang, Hai-Wen Feng, Jie Choi, Young-Jun Groleau, Denis Wang, Yi-Zhen Biomed Res Int Research Article OG2 is a modified antimicrobial peptide, that is, derived from the frog peptide Palustrin-OG1. It has high antimicrobial activity and low cytotoxicity, and it is therefore promising as a therapeutic agent. Both prokaryotic (Escherichia coli) and eukaryotic (Pichia pastoris) production host systems were used to produce OG2 in our previous study; however, it was difficult to achieve high expression yields and efficient purification. In this study, we achieved high-yield OG2 expression using the intein fusion system. The optimized OG2 gene was cloned into the pTWIN1 vector to generate pTWIN-OG2-intein2 (C-terminal fusion vector) and pTWIN-intein1-OG2 (N-terminal fusion vector). Nearly 70% of the expressed OG2-intein2 was soluble after the IPTG concentration and induction temperature were decreased, whereas only 42% of the expressed of intein1-OG2 was soluble. Up to 75 mg of OG2-intein2 was obtained from a 1 l culture, and 85% of the protein was cleaved by 100 mM DTT. Intein1-OG2 was less amenable to cleavage due to the inhibition of cleavage by the N-terminal amino acid of OG2. The purified OG2 exhibited strong antimicrobial activity against E. coli K88. The intein system is the best currently available system for the cost-effective production of OG2. Hindawi Publishing Corporation 2013 2013-07-02 /pmc/articles/PMC3713655/ /pubmed/23936842 http://dx.doi.org/10.1155/2013/754319 Text en Copyright © 2013 Yong-Gang Xie et al. https://creativecommons.org/licenses/by/3.0/ This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Xie, Yong-Gang
Han, Fei-Fei
Luan, Chao
Zhang, Hai-Wen
Feng, Jie
Choi, Young-Jun
Groleau, Denis
Wang, Yi-Zhen
High-Yield Soluble Expression and Simple Purification of the Antimicrobial Peptide OG2 Using the Intein System in Escherichia coli
title High-Yield Soluble Expression and Simple Purification of the Antimicrobial Peptide OG2 Using the Intein System in Escherichia coli
title_full High-Yield Soluble Expression and Simple Purification of the Antimicrobial Peptide OG2 Using the Intein System in Escherichia coli
title_fullStr High-Yield Soluble Expression and Simple Purification of the Antimicrobial Peptide OG2 Using the Intein System in Escherichia coli
title_full_unstemmed High-Yield Soluble Expression and Simple Purification of the Antimicrobial Peptide OG2 Using the Intein System in Escherichia coli
title_short High-Yield Soluble Expression and Simple Purification of the Antimicrobial Peptide OG2 Using the Intein System in Escherichia coli
title_sort high-yield soluble expression and simple purification of the antimicrobial peptide og2 using the intein system in escherichia coli
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3713655/
https://www.ncbi.nlm.nih.gov/pubmed/23936842
http://dx.doi.org/10.1155/2013/754319
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