Expression and purification of integral membrane metallopeptidase HtpX.
Little 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.
Proteolysis Lab, Department of Structural Biology, Molecular Biology Institute of Barcelona, CSIC, Barcelona Science Park, E-08028 Barcelona, Spain. Electronic address: jlacri@ibmb.csic.es.
Joan L
JL
Arolas
Proteolysis Lab, Department of Structural Biology, Molecular Biology Institute of Barcelona, CSIC, Barcelona Science Park, E-08028 Barcelona, Spain.
Raquel
R
García-Castellanos
Proteolysis Lab, Department of Structural Biology, Molecular Biology Institute of Barcelona, CSIC, Barcelona Science Park, E-08028 Barcelona, Spain.
Theodoros
T
Goulas
Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan.
Yoshinori
Y
Akiyama
Proteolysis Lab, Department of Structural Biology, Molecular Biology Institute of Barcelona, CSIC, Barcelona Science Park, E-08028 Barcelona, Spain. Electronic address: xgrcri@ibmb.csic.es.
F Xavier
FX
Gomis-Rüth
D003001:Cloning, Molecular; D003460:Crystallization; D003902:Detergents; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000302:isolation & purification*; D005960:Glucosides; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000302:isolation & purification*; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification*; D045726:Metalloproteases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification
D003902:Detergents; D029968:Escherichia coli Proteins; D005960:Glucosides; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C068607:HtpX protein, E coli; C018619:octyl-beta-D-glucoside; D045726:Metalloproteases
Escherichia coli; Integral membrane protein; Metallopeptidase; Overexpression; Protein quality control