Expression, purification and thermal stability evaluation of an engineered amaranth protein expressed in Escherichia coli

Background: The acidic subunit of amarantin (AAC)-the predominant amaranth seed storage protein-has functional potential and its third variable region (VR) has been modified with antihypertensive peptides to improve this potential. Here, we modified the C-terminal in the fourth VR of AAC by insertin...

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Autores principales:	Morales-Camacho,Jocksan I, Paredes-López,Octavio, Espinosa-Hernández,Edgar, Fernández Velasco,Daniel Alejandro, Luna-Suárez,Silvia
Lenguaje:	English
Publicado:	Pontificia Universidad Católica de Valparaíso 2016
Materias:	Globulin 11S Protein expression Protein engineering Thermal stability
Acceso en línea:	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582016000400007
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spelling	oai:scielo:S0717-345820160004000072016-09-13Expression, purification and thermal stability evaluation of an engineered amaranth protein expressed in Escherichia coliMorales-Camacho,Jocksan IParedes-López,OctavioEspinosa-Hernández,EdgarFernández Velasco,Daniel AlejandroLuna-Suárez,Silvia Globulin 11S Protein expression Protein engineering Thermal stability Background: The acidic subunit of amarantin (AAC)-the predominant amaranth seed storage protein-has functional potential and its third variable region (VR) has been modified with antihypertensive peptides to improve this potential. Here, we modified the C-terminal in the fourth VR of AAC by inserting four VY antihypertensive peptides. This modified protein (AACM.4) was expressed in Escherichia coli. In addition, we also recombinantly expressed other derivatives of the amarantin protein. These include: unmodified amarantin acidic subunit (AAC); amarantin acidic subunit modified at the third VR with four VY peptides (AACM.3); and amarantin acidic subunit doubly modified, in the third VR with four VY peptides and in the fourth VR with the RIPP peptide (AACM.3.4). Results: E. coli BL21-CodonPlus (DE3)-RIL was the most favorable strain for the expression of proteins. After 6 h of induction, it showed the best recombinant protein titer. The AAC and AACM.4 were obtained at higher titers (0.56 g/L) while proteins modified in the third VR showed lower titers: 0.44 g/L and 0.33 g/L for AACM.3 and AACM.3.4, respectively. As these AAC variants were mostly expressed in an insoluble form, we applied a refolding protocol. This made it possible to obtain all proteins in soluble form. Modification of the VR 4 improves the thermal stability of amarantin acidic subunit; AAC manifested melting temperature (Tm) at 34°C and AACM.4 at 37.2°C. The AACM.3 and AACM.3.4 did not show transition curves. Conclusions: Modifications to the third VR affect the thermal stability of amarantin acidic subunit.info:eu-repo/semantics/openAccessPontificia Universidad Católica de ValparaísoElectronic Journal of Biotechnology v.19 n.4 20162016-07-01text/htmlhttp://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582016000400007en10.1016/j.ejbt.2016.04.001
institution	Scielo Chile
collection	Scielo Chile
language	English
topic	Globulin 11S Protein expression Protein engineering Thermal stability
spellingShingle	Globulin 11S Protein expression Protein engineering Thermal stability Morales-Camacho,Jocksan I Paredes-López,Octavio Espinosa-Hernández,Edgar Fernández Velasco,Daniel Alejandro Luna-Suárez,Silvia Expression, purification and thermal stability evaluation of an engineered amaranth protein expressed in Escherichia coli
description	Background: The acidic subunit of amarantin (AAC)-the predominant amaranth seed storage protein-has functional potential and its third variable region (VR) has been modified with antihypertensive peptides to improve this potential. Here, we modified the C-terminal in the fourth VR of AAC by inserting four VY antihypertensive peptides. This modified protein (AACM.4) was expressed in Escherichia coli. In addition, we also recombinantly expressed other derivatives of the amarantin protein. These include: unmodified amarantin acidic subunit (AAC); amarantin acidic subunit modified at the third VR with four VY peptides (AACM.3); and amarantin acidic subunit doubly modified, in the third VR with four VY peptides and in the fourth VR with the RIPP peptide (AACM.3.4). Results: E. coli BL21-CodonPlus (DE3)-RIL was the most favorable strain for the expression of proteins. After 6 h of induction, it showed the best recombinant protein titer. The AAC and AACM.4 were obtained at higher titers (0.56 g/L) while proteins modified in the third VR showed lower titers: 0.44 g/L and 0.33 g/L for AACM.3 and AACM.3.4, respectively. As these AAC variants were mostly expressed in an insoluble form, we applied a refolding protocol. This made it possible to obtain all proteins in soluble form. Modification of the VR 4 improves the thermal stability of amarantin acidic subunit; AAC manifested melting temperature (Tm) at 34°C and AACM.4 at 37.2°C. The AACM.3 and AACM.3.4 did not show transition curves. Conclusions: Modifications to the third VR affect the thermal stability of amarantin acidic subunit.
author	Morales-Camacho,Jocksan I Paredes-López,Octavio Espinosa-Hernández,Edgar Fernández Velasco,Daniel Alejandro Luna-Suárez,Silvia
author_facet	Morales-Camacho,Jocksan I Paredes-López,Octavio Espinosa-Hernández,Edgar Fernández Velasco,Daniel Alejandro Luna-Suárez,Silvia
author_sort	Morales-Camacho,Jocksan I
title	Expression, purification and thermal stability evaluation of an engineered amaranth protein expressed in Escherichia coli
title_short	Expression, purification and thermal stability evaluation of an engineered amaranth protein expressed in Escherichia coli
title_full	Expression, purification and thermal stability evaluation of an engineered amaranth protein expressed in Escherichia coli
title_fullStr	Expression, purification and thermal stability evaluation of an engineered amaranth protein expressed in Escherichia coli
title_full_unstemmed	Expression, purification and thermal stability evaluation of an engineered amaranth protein expressed in Escherichia coli
title_sort	expression, purification and thermal stability evaluation of an engineered amaranth protein expressed in escherichia coli
publisher	Pontificia Universidad Católica de Valparaíso
publishDate	2016
url	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582016000400007
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Expression, purification and thermal stability evaluation of an engineered amaranth protein expressed in Escherichia coli

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