English   español  
Por favor, use este identificador para citar o enlazar a este item: http://hdl.handle.net/10261/101844
COMPARTIR / IMPACTO:
Estadísticas
logo share SHARE logo core CORE   Add this article to your Mendeley library MendeleyBASE

Visualizar otros formatos: MARC | Dublin Core | RDF | ORE | MODS | METS | DIDL
Exportar a otros formatos:
Título

Expression and purification of integral membrane metallopeptidase HtpX

AutorArolas, Joan L.; García-Castellanos, Raquel; Goulas, Theodoros ; Akiyama, Yoshinori; Gomis-Rüth, F. Xavier
Palabras claveMetallopeptidase
Integral membrane protein
Escherichia coli
Overexpression
Protein quality control
Fecha de publicación2014
EditorAcademic Press
CitaciónProtein Expression and Purification 99: 113-118 (2014)
ResumenLittle is known about the catalytic mechanism of integral membrane (IM) peptidases. HtpX is an IM metallopeptidase that plays a central role in protein quality control by preventing the accumulation of misfolded proteins in the membrane. Here we report the recombinant overexpression and purification of a catalytically ablated form of HtpX from Escherichia coli. Several E. coli strains, expression vectors, detergents, and purification strategies were tested to achieve maximum yields of pure and well-folded protein. HtpX was successfully overexpressed in E. coli BL21(DE3) cells using a pET-derived vector attaching a C-terminal His8-tag, extracted from the membranes using octyl-β-d-glucoside, and purified to homogeneity in the presence of this detergent in three consecutive steps: cobalt-affinity, anion-exchange, and size-exclusion chromatography. The production of HtpX in milligram amounts paves the way for structural studies, which will be essential to understand the catalytic mechanism of this IM peptidase and related family members. © 2014 Elsevier Inc. All rights reserved.
Versión del editorhttp://dx.doi.org/10.1016/j.pep.2014.04.008
URIhttp://hdl.handle.net/10261/101844
DOI10.1016/j.pep.2014.04.008
Identificadoresdoi: 10.1016/j.pep.2014.04.008
issn: 1046-5928
Aparece en las colecciones: (IBMB) Artículos
Ficheros en este ítem:
Fichero Descripción Tamaño Formato  
Arolas_PEP_2014_corrMs.pdf789,54 kBAdobe PDFVista previa
Visualizar/Abrir
Mostrar el registro completo
 

Artículos relacionados:


NOTA: Los ítems de Digital.CSIC están protegidos por copyright, con todos los derechos reservados, a menos que se indique lo contrario.