Reversible biotinylation of C1q with a cleavable biotinyl derivative. Application in C1q receptor (C1qR) purification.
Reversible biotinylation of human C1q without impairment of its physiologic functions has allowed us to develop a simple and rapid purification method for C1q receptor (C1qR). The biotinylating reagent, NHS-SS-biotin (Mr 606.7) contains an extended connector or cross-linker arm which limits steric hindrance and is bridged by a cleavable disulfide bond to the biotin component. Biotinylation was achieved by mixing C1q (in PBS, pH 7.4) with NHS-SS-biotin (dissolved in dimethyl formamide) in a 50:1 v/v and 1:25 mol/mol ratio and allowing the reaction to continue at room temperature for 4 h. The mixture was then dialyzed against PBS pH 7.4 (2 X 1 liter) and analyzed by SDS-PAGE and hemolytic assay using C1q depleted serum. Under these conditions neither denaturation of the protein nor loss of hemolytic activity was evident. Such biotinylated C1q (Bio-C1q) was used to pull out the C1qR from detergent-solubilized (1% NP-40 in PBS, pH 7.4 plus inhibitors) 125I-surface labeled membrane solution that had been first centrifuged (1 h, 45,000 X g, 4 degrees C) and then sequentially precleared with immobilized protein A, protein A-IgG and gelatin. The mixture of Bio-C1q and membrane solution was then incubated (20 h, 4 degrees C), applied to immobilized avidin (equilibrated with PBS, pH 7.4, 0.1% NP-40) and after washing, the bound C1qR was eluted with equilibrating buffer containing 1 M NaCl, and the C1q by same buffer containing 100 mM DTT. The eluted C1qR contained a major Mr 70,000 molecule which upon reduction electrophoresed with an apparent Mr of 85,000-90,000 as assessed by SDS-PAGE analysis. In addition, a faint single chain band of 30-40 kDa was eluted with the major band and may represent a non-covalently associated part of the C1qR molecule.
Ghebrehiwet
B
B
Department of Medicine, State University of New York, Stony Brook 11794-8161.
Bossone
S
S
Erdei
A
A
Reid
K B
KB
D001360:Avidin; D001710:Biotin / Q000031:analogs & derivatives*; D002352:Carrier Proteins; D003166:Complement Activating Enzymes / Q000378:metabolism*; D003172:Complement C1 / Q000378:metabolism*; D015922:Complement C1q; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D004800:Enzymes, Immobilized; D006461:Hemolysis; D006801:Humans; D018960:Hyaluronan Receptors; D007122:Immunoelectrophoresis; D008562:Membrane Glycoproteins; D024101:Mitochondrial Proteins; D011951:Receptors, Complement / Q000276:immunology / Q000302:isolation & purification*; D013329:Structure-Activity Relationship; D013388:Succinimides
C087299:C1QBP protein, human; D002352:Carrier Proteins; D003172:Complement C1; D004220:Disulfides; D004800:Enzymes, Immobilized; D018960:Hyaluronan Receptors; D008562:Membrane Glycoproteins; D024101:Mitochondrial Proteins; D011951:Receptors, Complement; D013388:Succinimides; C033788:biotinyl N-hydroxysuccinimide ester; C032402:complement 1q receptor; D001360:Avidin; D001710:Biotin; D015922:Complement C1q; D003166:Complement Activating Enzymes