Functional refolding of a recombinant C-type lectin-like domain containing intramolecular disulfide bonds. Export

@article{citeulike:5776342, abstract = {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.}, author = {Vohra, Ravinder S. and Murphy, Jane E. and Walker, John H. and Homer-Vanniasinkam, Shervanthi and Ponnambalam, Sreenivasan}, citeulike-article-id = {5776342}, citeulike-linkout-0 = {http://dx.doi.org/10.1016/j.pep.2006.11.012}, citeulike-linkout-1 = {http://view.ncbi.nlm.nih.gov/pubmed/17196395}, citeulike-linkout-2 = {http://www.hubmed.org/display.cgi?uids=17196395}, doi = {10.1016/j.pep.2006.11.012}, issn = {1046-5928}, journal = {Protein expression and purification}, keywords = {lox-1}, month = {April}, number = {2}, pages = {415--421}, posted-at = {2009-10-21 19:05:11}, priority = {2}, title = {Functional refolding of a recombinant C-type lectin-like domain containing intramolecular disulfide bonds.}, url = {http://dx.doi.org/10.1016/j.pep.2006.11.012}, volume = {52}, year = {2007} }

TY - JOUR ID - citeulike:5776342 L3 - citeulike-article-id:5776342 N2 - 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. IS - 2 JF - Protein expression and purification SN - 1046-5928 EP - 421 TI - Functional refolding of a recombinant C-type lectin-like domain containing intramolecular disulfide bonds. VL - 52 SP - 415 KW - lox-1 AU - Vohra, Ravinder AU - Murphy, Jane AU - Walker, John AU - Homer-Vanniasinkam, Shervanthi AU - Ponnambalam, Sreenivasan PY - 2007/04// UR - http://dx.doi.org/10.1016/j.pep.2006.11.012 ER -