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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.
Pubmed ID: 17196395
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It contains entries for refolding protocols for a wide range of proteins. REFOLD provides a means of dissminating refolding protocols and techniques to the scientific community. A large number of recombinant proteins expressed in bacteria are insoluble and thus require renaturation. Identifying optimal conditions and methodology for refolding can be time consuming and often rate-limiting. To this end, REFOLD was designed to assist in the design and implementation of methodologies for the in vitro refolding of proteins. The database contains heavily annotated entries for the refolding of a wide range of proteins which can be searched via multiple parameters using either simple or advanced search functions. The database can also be browsed by categories such as structural class, family, or refolding method. The web interface allows a detailed, sortable, spreadsheet-like list of results allowing quick visualisation of search results. Each entry contains detailed information regarding the protein of interest, methods and conditions employed, as well as reference and links to the relevant journal publication. Effective use of hyperlinks in results pages also allows useful browsing of entries for other proteins sharing similar properties or methods. REFOLD also provides analysis of the database through graphical representation of the data. The available graphs show the breakdown of refolding records in the database according to different parameters, such as refolding method, protein constructs and various refolding conditions. REFOLD graphs can be found here.
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