Purification of recombinant C-reactive protein mutants.
C-reactive protein (CRP) is an evolutionarily conserved protein, a component of the innate immune system, and an acute phase protein in humans. In addition to its raised level in blood in inflammatory states, CRP is also localized at sites of inflammation including atherosclerotic lesions, arthritic joints and amyloid plaque deposits. Results of in vivo experiments in animal models of inflammatory diseases indicate that CRP is an anti-pneumococcal, anti-atherosclerotic, anti-arthritic and an anti-amyloidogenic molecule. The mechanisms through which CRP functions in inflammatory diseases are not fully defined; however, the ligand recognition function of CRP in its native and non-native pentameric structural conformations and the complement-activating ability of ligand-complexed CRP have been suggested to play a role. One tool to understand the structure-function relationships of CRP and determine the contributions of the recognition and effector functions of CRP in host defense is to employ site-directed mutagenesis to create mutants for experimentation. For example, CRP mutants incapable of binding to phosphocholine are generated to investigate the importance of the phosphocholine-binding property of CRP in mediating host defense. Recombinant CRP mutants can be expressed in mammalian cells and, if expressed, can be purified from the cell culture media. While the methods to purify wild-type CRP are well established, different purification strategies are needed to purify various mutant forms of CRP if the mutant does not bind to either calcium or phosphocholine. In this article, we report the methods used to purify pentameric recombinant wild-type and mutant CRP expressed in and secreted by mammalian cells.
Department of Biomedical Sciences, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, United States.
Avinash
A
Thirumalai
Department of Biomedical Sciences, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, United States.
Sanjay K
SK
Singh
Department of Biomedical Sciences, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, United States.
David J
DJ
Hammond
Department of Biomedical Sciences, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, United States.
Toh B
TB
Gang
Department of Biomedical Sciences, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, United States.
Donald N
DN
Ngwa
Department of Biomedical Sciences, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, United States.
Asmita
A
Pathak
Department of Biomedical Sciences, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, United States. Electronic address: agrawal@etsu.edu.
Alok
A
Agrawal
D000818:Animals; D000837:Anion Exchange Resins / Q000737:chemistry; D001665:Binding Sites; D002097:C-Reactive Protein / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004983:Ethanolamines / Q000737:chemistry; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010767:Phosphorylcholine / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification; D013329:Structure-Activity Relationship; D014162:Transfection
D000837:Anion Exchange Resins; D004983:Ethanolamines; D011994:Recombinant Proteins; D010767:Phosphorylcholine; C005448:phosphorylethanolamine; D002097:C-Reactive Protein; D002118:Calcium
C-reactive protein; Phosphocholine; Phosphoethanolamine