Intein-mediated expression of cecropin in Escherichia coli

Different strategies have been used to overcome the difficulties to produce antimicrobial peptides. Here we used Intein Mediated Purification with an Affinity Chitin-binding Tag (IMPACT-System, New England Biolabs) for the expression of the antimicrobial peptide cecropin to reduce its sensitivity to...

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Autores principales:	Díaz,Mauricio, Venturini,Elena, Marchetti,Stefano, Arenas,Gloria, Marshall,Sergio H
Lenguaje:	English
Publicado:	Pontificia Universidad Católica de Valparaíso 2012
Materias:	antimicrobial cecropin fusion intein peptide soluble
Acceso en línea:	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582012000200003
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spelling	oai:scielo:S0717-345820120002000032012-06-06Intein-mediated expression of cecropin in Escherichia coliDíaz,MauricioVenturini,ElenaMarchetti,StefanoArenas,GloriaMarshall,Sergio H antimicrobial cecropin fusion intein peptide soluble Different strategies have been used to overcome the difficulties to produce antimicrobial peptides. Here we used Intein Mediated Purification with an Affinity Chitin-binding Tag (IMPACT-System, New England Biolabs) for the expression of the antimicrobial peptide cecropin to reduce its sensitivity to intracellular proteases and use its inducible self-cleaving capability to remove the carrier. Cecropin was cloned into suitable expression vector pTYB11, and expression induced by IPTG in Escherichia coli ER2566. The use of 22ºC induction allowed the expression of cecropin with its intein carrier in soluble form. Cell extracts were purified by chitin affinity chromatography and intein-mediated splicing of the target protein was achieved by thiol addition, obtaining a final yield of 2.5 mg cecropin/l. Cecropin cleaved from the intein had its proper biologically active form, showing a micromolar antimicrobial activity against Vibrio ordalii, Vibrio alginolyticus and Escherichia coli.info:eu-repo/semantics/openAccessPontificia Universidad Católica de ValparaísoElectronic Journal of Biotechnology v.15 n.2 20122012-03-01text/htmlhttp://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582012000200003en
institution	Scielo Chile
collection	Scielo Chile
language	English
topic	antimicrobial cecropin fusion intein peptide soluble
spellingShingle	antimicrobial cecropin fusion intein peptide soluble Díaz,Mauricio Venturini,Elena Marchetti,Stefano Arenas,Gloria Marshall,Sergio H Intein-mediated expression of cecropin in Escherichia coli
description	Different strategies have been used to overcome the difficulties to produce antimicrobial peptides. Here we used Intein Mediated Purification with an Affinity Chitin-binding Tag (IMPACT-System, New England Biolabs) for the expression of the antimicrobial peptide cecropin to reduce its sensitivity to intracellular proteases and use its inducible self-cleaving capability to remove the carrier. Cecropin was cloned into suitable expression vector pTYB11, and expression induced by IPTG in Escherichia coli ER2566. The use of 22ºC induction allowed the expression of cecropin with its intein carrier in soluble form. Cell extracts were purified by chitin affinity chromatography and intein-mediated splicing of the target protein was achieved by thiol addition, obtaining a final yield of 2.5 mg cecropin/l. Cecropin cleaved from the intein had its proper biologically active form, showing a micromolar antimicrobial activity against Vibrio ordalii, Vibrio alginolyticus and Escherichia coli.
author	Díaz,Mauricio Venturini,Elena Marchetti,Stefano Arenas,Gloria Marshall,Sergio H
author_facet	Díaz,Mauricio Venturini,Elena Marchetti,Stefano Arenas,Gloria Marshall,Sergio H
author_sort	Díaz,Mauricio
title	Intein-mediated expression of cecropin in Escherichia coli
title_short	Intein-mediated expression of cecropin in Escherichia coli
title_full	Intein-mediated expression of cecropin in Escherichia coli
title_fullStr	Intein-mediated expression of cecropin in Escherichia coli
title_full_unstemmed	Intein-mediated expression of cecropin in Escherichia coli
title_sort	intein-mediated expression of cecropin in escherichia coli
publisher	Pontificia Universidad Católica de Valparaíso
publishDate	2012
url	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582012000200003
work_keys_str_mv	AT diazmauricio inteinmediatedexpressionofcecropininescherichiacoli AT venturinielena inteinmediatedexpressionofcecropininescherichiacoli AT marchettistefano inteinmediatedexpressionofcecropininescherichiacoli AT arenasgloria inteinmediatedexpressionofcecropininescherichiacoli AT marshallsergioh inteinmediatedexpressionofcecropininescherichiacoli
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Intein-mediated expression of cecropin in Escherichia coli

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