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Título

Expression and purification of integral membrane metallopeptidase HtpX

Autor Arolas, Joan L.; García-Castellanos, Raquel; Goulas, Theodoros ; Akiyama, Yoshinori; Gomis-Rüth, F. Xavier
Palabras clave Metallopeptidase
Integral membrane protein
Escherichia coli
Overexpression
Protein quality control
Fecha de publicación 2014
EditorAcademic Press
Citación Protein 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
URI http://hdl.handle.net/10261/101844
DOI10.1016/j.pep.2014.04.008
Identificadoresdoi: 10.1016/j.pep.2014.04.008
issn: 1046-5928
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