A simplified procedure for C-reactive protein purification

Abstract

A simplified procedure for obtaining high purity and high yield of C-reactive protein (CRP) was established. The CRP from ascitic and pleural fluid was purified using calcium dependent affinity chromatography of CNBr-activated Sepharose-4B covalently coupled to p-diazonium phenylphosphorylcholine (DPPC) and hydroxylapatite chromatography. The active fractions of CRP during the purification procedure were determined by EIA using pneumococcal C-polysaccharide. The final recovery of CRP was 67% of the initial protein amount as quantitated by single radial immunodiffusion (SRID), and the purification fold was 3,600. The purified CRP was shown to be homogeneous by electrophoresis and immunoelectrophoresis. The molecular weight of CRP was approximately 118’KD as determined by Sephacryl S-200 gel filtration and the molecular weight of a subunit was approximately 23.6 KD by SDS-polyacrylamide gel electrophoresis. The isoelectric point of CRP was found to range about 4.82.