Affinity purification of a mannose-binding protein, a sensitive tool in the diagnostics of IgM, via site-directed phosphorylated mannan bound to alumina.
Ca2+ -dependent mannose-binding proteins (MBPs) belong to the family of animal lectins. They perform in vivo as defence molecules that act as opsonins by enhancing the clearance of mannose rich pathogens and have been used in vitro for the purification of IgM. MBPs have been previously isolated by methods based on binding the protein moiety of various mannan species to different matrices. However, the mannan-protein complexes did not have a constant protein content and the yield of the isolated MBPs was variable. In the present study we describe a new approach for the affinity purification of MBPs based on the main polysaccharide moiety of the complex. After removal of residual phosphate groups naturally occurring at the C-3 position of the sugar, which interfere with MBP recognition, the mannan was phosphorylated enzymatically at C-6, at which position the OH group is not required for lectin binding. The enzymatically phosphorylated mannan bound to an alumina column was used successfully for MBP separation from rabbit serum. The mannose-binding protein obtained was used in our study for diagnostic purposes in the identification and determination of very low concentrations of IgM.
Koppel
R
R
Department of Molecular Microbiology and Biotechnology, Tel-Aviv University, Ramat-Aviv, Israel.
Litvak
M
M
Solomon
B
B
D000537:Aluminum Oxide; D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification*; D004797:Enzyme-Linked Immunosorbent Assay; D007074:Immunoglobulin G / Q000032:analysis; D007075:Immunoglobulin M / Q000032:analysis*; D008351:Mannans / Q000737:chemistry; D008358:Mannose / Q000737:chemistry*; D037241:Mannose-Binding Lectins; D010766:Phosphorylation; D011817:Rabbits
D002352:Carrier Proteins; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D008351:Mannans; D037241:Mannose-Binding Lectins; D000537:Aluminum Oxide; D008358:Mannose