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Rapid purification of the human C3b/C4b receptor (CR1) by monoclonal antibody affinity chromatography |
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| Authors: | W W Wong R M Jack J A Smith C A Kennedy D T Fearon |
| Affiliation: | 1. Brigham and Women''s Hospital, Department of Rheumatology and Immunology, and Harvard Medical School, Department of Medicine, 607 Seeley G. Mudd Building, 250 Longwood Avenue, Boston, MA 02115, U.S.A.;2. Massachusetts General Hospital, Departments of Molecular Biology and Pathology, and Harvard Medical School, Department of Pathology, 50 Blossom Street, Boston, MA 02114, U.S.A. |
| Abstract: | The human C3b/C4b receptor (CR1) is a polymorphic glycoprotein that is expressed on erythrocytes, leukocytes and glomerular podocytes. Further structural analysis and molecular genetic studies would be facilitated by the availability of relatively larger amounts of purified CR1. Milligram quantities of CR1 were purified from erythrocyte membranes 10,000-fold with an average yield of 30-40% by a rapid procedure which utilized sequential chromatography on Matrex Red A and a monoclonal anti-CR1 antibody affinity column. The purified receptor was homogeneous by SDS-PAGE and consisted of the 2 most common alleles of CR1. Purified CR1 also retained its function of serving as a cofactor for the cleavage of C3b to iC3b, C3dg and C3c. The amino acid composition was typical of that of a globular protein and sequence analysis of the N-terminus of the purified CR1 revealed that it was blocked. |
| Keywords: | human CR1 purification C3 monoclonal antibody amino acid composition CR1 the human C3b/C4b receptor EDTA ethylenediamine tetraacetate DFP diisopropylfluorophosphate DOC deoxycholic acid I C3b/C4b inactivator NP-40 Nonidet P-40 PAGE polyacrylamide gel electrophoresis PBS 10 mM sodium phosphate, 150 mM NaCl, pH 7.4 PBNS PBS containing 1% NP-40 and 0.2% SDS PMSF phenylmethylsulfonylfluoride Pth 3-phenyl-2-thiohydantoin SDS sodium dodecyl sulfate |
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