Vohra, RS; Murphy, JE; Walker, JH; Homer-Vanniasinkam, S; Ponnambalam, S; (2007) Functional refolding of a recombinant C-type lectin-like domain containing intramolecular disulfide bonds. PROTEIN EXPRES PURIF , 52 (2) 415 - 421. 10.1016/j.pep.2006.11.012.
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The lectin-like oxidized low-density lipoprotein scavenger receptor (LOX-1) is a pro-inflammatory marker and Type 11 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. (c) 2006 Elsevier Inc. All rights reserved.
|Title:||Functional refolding of a recombinant C-type lectin-like domain containing intramolecular disulfide bonds|
|Keywords:||LOX-1, refolding, oxLDL, LOW-DENSITY-LIPOPROTEIN, LIGAND-BINDING DOMAIN, CRYSTAL-STRUCTURE, SCAVENGER-RECEPTOR, LDL-RECEPTOR, LOX-1, CELLS, ATHEROSCLEROSIS, RECOGNITION, EXPRESSION|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Medical Sciences > Surgery and Interventional Science (Division of)|
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