One-step on-column affinity refolding purification and functional analysis of recombinant human VDAC1.
The outer mitochondrial membrane porin, voltage-dependent anion-selective channel (VDAC), is believed to play an important role in mediating mitochondria-dependent apoptosis. However, detailed structure-function studies of VDAC have been hindered by the difficulties to obtain a soluble, correctly folded, and fully active form of the recombinant VDAC and its mutant variants due to its transmembrane nature. Here we report a high-throughput one-step chromatographic procedure in purification of recombinant human VDAC1 (rhVDAC1) protein overexpressed in bacteria. The improved methodology could generate a large quantity of rhVDAC1 with correct folding in terms of the secondary structure, with full biological activities in mediating cytochrome c release and in interaction with Bcl-X(L). The method will significantly benefit genetic, biochemical, and structural studies of this critical channel protein.
Shi
Yong
Y
The Center for Molecular Microbiology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China.
Jiang
Chunsun
C
Chen
Quan
Q
Tang
Hong
H
D002846:Chromatography, Affinity / Q000379:methods; D002942:Circular Dichroism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007473:Ion Channels / Q000737:chemistry / Q000302:isolation & purification; D046911:Macromolecular Substances; D018272:Porins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D050995:Voltage-Dependent Anion Channel 1; D050994:Voltage-Dependent Anion Channels; D051020:bcl-X Protein
C494811:BCL2L1 protein, human; D007473:Ion Channels; D046911:Macromolecular Substances; D018272:Porins; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C494762:VDAC1 protein, human; D050994:Voltage-Dependent Anion Channels; D051020:bcl-X Protein; D050995:Voltage-Dependent Anion Channel 1