Functional refolding of a recombinant C-type lectin-like domain containing intramolecular disulfide bonds.
The lectin-like oxidized low-density lipoprotein scavenger receptor (LOX-1) is a pro-inflammatory marker and Type II membrane protein expressed on vascular cells and tissues. The LOX-1 extracellular domain mediates recognition of oxidized low-density lipoprotein (oxLDL) particles that are implicated in the development of atherosclerotic plaques. To study the molecular basis for LOX-1-mediated ligand recognition, we have expressed, purified and refolded a recombinant LOX-1 protein and assayed for its biological activity using a novel fluorescence-based assay to monitor binding to lipid particles. Overexpression of a hexahistidine-tagged cysteine-rich LOX-1 extracellular domain in bacteria leads to the formation of aggregates that accumulated in bacterial inclusion bodies. The hexahistidine-tagged LOX-1 molecule was purified by affinity chromatography from solubilized inclusion bodies. A sequential dialysis procedure was used to refold the purified but inactive and denatured LOX-1 protein into a functionally active form that mediated recognition of oxLDL particles. This approach allowed slow LOX-1 refolding and assembly of correct intrachain disulfide bonds. Circular dichroism analysis of the refolded LOX-1 molecule demonstrated a folded state with substantial alpha-helical content. Using immobilized recombinant, refolded LOX-1 we demonstrated a 70-fold preferential recognition for oxLDL over native LDL particles. Thus, a protein domain containing intrachain disulfide bonds can be reconstituted into a functionally active state using a relatively simple dialysis-based technique.
Vohra
Ravinder S
RS
Endothelial Cell Biology Unit, Leeds Institute of Genetics, Health and Therapeutics, University of Leeds, Leeds LS2 9JT, UK.
Murphy
Jane E
JE
Walker
John H
JH
Homer-Vanniasinkam
Shervanthi
S
Ponnambalam
Sreenivasan
S
D004220:Disulfides / Q000737:chemistry*; D008077:Lipoproteins, LDL / Q000378:metabolism*; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D051127:Scavenger Receptors, Class E / Q000737:chemistry / Q000378:metabolism*
D004220:Disulfides; D008077:Lipoproteins, LDL; C495137:OLR1 protein, human; D011994:Recombinant Proteins; D051127:Scavenger Receptors, Class E; C099252:oxidized low density lipoprotein