Rapid isolation and characterization of native mouse complement components C3 and C5

Abstract

A rapid, 1 day procedure for the purification of mouse complement factors C3 and C5 is described. The method is based on fractionated precipitation by polyethylene glycol 6000, followed by Mono Q anion exchange chromatography on a system for fast protein liquid chromatography (FPLC). For C3 isolation, an additional FPLC separation step using Superose 12 (gel filtration) was used. C3 was purified 71-fold with a yield of 32% as measured by biological activity; the preparation contained no detectable contaminants as judged by SDS-PAGE. A comparable procedure for the isolation of C5 resulted in a preparation with a considerable contamination which could be easily removed by affinity chromatography using antibodies directed against these contaminants. With this combined procedure C5 was purified 536-fold with a yield of 28% based on biological activity. SDS-polyacrylamide gel electrophoresis revealed that mouse C3 and C5 had apparent M ss of 170 000 and 190 000, respectively. Under reducing conditions the α and β chains showed M rs of 107 000 and 62 000 for C3, and 104 000 and 85 000 for C5.