Solubilization and purification of recombinant modified C-reactive protein from inclusion bodies using reversible anhydride modification

摘要

The precise function of C-reactive protein（CRP） as a regulator of inflammation in health and disease continues to evolve. The true understanding of its role in host defense responses has been hampered by numerous reports of comparable systems with contradictory interpretations of CRP as a stimulator, suppressor, or benign contributor to such processes. These discrepancies may be explained in part by the existence of a naturally occurring CRP isoform, termed modified CRP（i.e., m CRP）, that is expressed when CRP subunits are dissociated into monomeric structures. The free m CRP subunit undergoes a non-proteolytic conformational change that has unique solubility, antigenicity, and bioactivity compared to the subunits that remain associated in the native, pentameric CRP molecule（i.e., p CRP）. As specific reagents have been developed to identify and quantify m CRP, it has become apparent that this isoform can be formed spontaneously in calcium-free solutions. Furthermore, m CRP can be expressed on perturbed cell membranes with as little as 24–48 h incubation in tissue culture. Because m CRP has the same size as p CRP subunits as evaluated by SDS-PAGE, its presence in a p CRP reagent would not be apparent using this technique to evaluate purity. Finally, because many antibody reagents purported to be specific for ‘‘CRP’contains some, or substantial specificity to m CRP, antigen-detection techniques using such reagents may fail to distinguish the specific CRP isoform detected. All these caveats concerning CRP structures and measurements suggest that the aforementioned contradictory studies may reflect to some extent on distinctive bioactivities of m CRP rather than on p CRP. To provide a reliable, abundant supply of m CRP for separate and comparable studies, a recombinant protein was engineered and expressed in E. coli（i.e., recombinant m CRP or rmCRP）. Synthesized protein was produced as inclusion bodies which proved difficult to solubilize for purification and characterization. Herein, we describe a method using anhydride reagents to effectively solubilize rmCRP and allow for chromatographic purification in high yield and free of contaminating endotoxin. Furthermore, the purified rmCRP reagent represents an excellent comparable protein to the biologically produced m CRP and as a distinctive reagent from p CRP. Deciphering the true function of CRP in both health and disease requires a knowledge, understanding, and reliable supply of each of its structures so to define the distinctive effects of each on the body’s response to tissue damaging events.