Reversible biotinylation of C1q with a cleavable biotinyl derivative: Application in C1q receptor (C1qR) purification

Abstract

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 ( M r 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 × 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 × g, 4°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°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 M r 70 000 molecule which upon reduction electrophoresed with an apparent M r 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.