Purified C5a receptor from human polymorphonuclear leukocytes
AssigneeMerck & Co., Inc.
InventorsThomas E. Rollins, Martin S. Springer
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Abstract
A method is disclosed for isolating and purifying C5a receptor from human polymorphonuclear leukocytes. C5a is a complement-derived protein which is important as a mediator of inflammatory responses. C5a receptor may be used to screen create and quantify C5a antagonists useful as anti-inflammatory agents and immunoregulants and to generate monoclonal and polyclonal anti-C5a receptor antibodies useful as anti-inflammatory agents and immunoregulants.
Claims6
A C5a receptor, the receptor having a K.sub.d of binding C5a approximately equal to that of C5a receptor on intact cells, having a molecular mass of 42 kDa when determined by SDS-PAGE under reducing conditions;
- and containing less than 10% other cellular matter and plasma with the proviso that the preparation need not be free of one or more associated G-proteins.
A highly purified and concentrated C5a receptor having the activity of binding C5a, said C5a receptor prepared by the steps comprising:
- (a) solubilizing an impure preparation selected from the group consisting of polymorphonuclear leukocytes, macrophage, eosinophil, ATCC U937 and HL60, said solubilizing comprising, addition of a solution buffered at pH 6 to 8 with 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, said solution containing approximately 0.5 to 2.0% of a first detergent which is digitonin, to the impure preparation;
- (b) contacting the impure preparation with a matrix to which an agent specfic for C5a binding activity has been bound;
- (c) adsorbing from the impure preparation, C5a receptor having C5a binding activity onto said matrix;
- (d) removing the impurities from the C5a receptor bound to said agent;
- and
- (e) desorbing the C5a receptor from said matrix, said desorbing step comprising, eluting the C5a receptor with a solution buffered at pH 4.0 to 5.0, said solution containing 0.05% to 0.5% of a second detergent which is digitonin, to the impure preparation;
- and 0.1 to 0.4M of a chaotrope.
Dependencies are inferred from the claim text.