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Purification and characterization of bovine mannan-binding protein |
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| Authors: | Holmskov, Uffe Holt, Palle Reid, Kenneth B.M. Willis, Anthony C. Teisner, Borge Jensenius, Jens Christian |
| Affiliation: | 1Department of Medical Microbiology, Institute of Medical Biology, University of Odense DK-5000 Odense C, Denmark 2MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford South Parks Road, Oxford, OX1 3QU, UK 3Division of Immunology, The State Serum Institute DK-2300, Copenhagen 4Department of Immunology, Institute of Medical Microbiology, University of Aarhus DK-8000 Aarhus C, Denmark |
| Abstract: | Bovine mannan-binding protein (bMBP) was observed in serum byits Ca2+ -dependent binding to mannan and by an Mrof 28 kDaunder reducing conditions on sodium dodecyl sulphatepolyacrylamidegel electrophoresis (SDSPAGE). The lectin was isolatedby precipitation with polyethyl-eneglycol (PEG), affinity chromatographyon mannanSepharose eluted with EDTA, and absorption onSepharose 4B rabbit anti-bovine Ig to remove anti-mannan antibodies.Fractions containing the lectin were reapplied to mannanSepharoseand eluted first with N-acetyl-D-glucosamine (GlcNAc) to removeconglutinin, and then with mannose to elute the 28 kDa lectin.Further purification was achieved by ion-exchange chromatographyon Mono-Q and by man-nose-gradient elution from a mannan-Sepharosecolumn. SDSPAGE of the purified lectin showed three highmolecular weight bands under non-reducing conditions. The reducedprotein gave a single band of 28 kDa. On gel permeation chromatographyunder non-dissociating conditions, the protein emerged at avolume corresponding to Mr |
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