Purification of Recombinant Mouse C-Reactive Protein fromPichia PastorisGS115 by Nickel Chelating Sepharose Fast-Flow Affinity Chromatography andP-Aminophenyl Phosphoryl Choline Agarose Resin Affinity Chromatography in Tandem

Abstract

C-reactive protein (CRP) is a circulating marker of inflammation yet with ill-defined biological functions. This is partly due to the uncharacterized activities of endogenous CRP in mice, the major animal model used to define protein function. The hurdles for purification and characterization of mouse CRP are its low circulating levels and the lack of specific antibodies. To clear these hurdles, here we developed an efficient expression system by constructing recombinant Pichia pastoris cells for secretion of native conformation mouse CRP. The recombinant expression of mouse CRP in Escherichia coli failed to yield sufficient amount of native protein, reflecting the importance of post-translational modification of glycosylation in aiding proper folding. By contrast, sufficient amount of native mouse CRP was successfully purified from P. pastoris. Preliminary purification was performed by Nickel Chelating Sepharose Fast-Flow affinity chromatography with 6 × His tags attached to the protein. Subsequently, p-Aminophenyl Phosphoryl Choline Agarose resin affinity chromatography was used for tandem purification. The purified mouse CRP showed native pentamer and capabilities of PC binding. Moreover, the 6 × His tag provides a convenient tool for detecting the interactions of mouse CRP with ligands.

C-reactive protein: structure, function, regulation, and role in clinical diseases

“…Many studies have used calcium-dependent PC affinity to successfully separate CRP from blood, such as horseshoe crab ( 32 ), mouse ( 33 ), dog ( 34 ), cat ( 35 ), cow ( 36 ), since the calcium-dependent PC affinity is conserved in the species and as a basis for testing the function of multi-species CRP in experiments. The CRP of human, mouse and rat were expressed in different systems and purified based on the calcium-dependent binding properties to PC ( 37 – 40 ). The binding ability of pentamer CRP to pattern recognition ligand PC was no significant difference among different species ( 38 ).…”