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

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Biophysics Reports 2015年第1期1卷 18-33页

作者： lawrence a.potempa Zhen-Yu Yao Shang-Rong Ji János G.Filep Yi Wu Roosevelt University College of Pharmacy MOE Key Laboratory of Cell Activities and Stress Adaptations School of Life SciencesLanzhou University Research Center Maisonneuve-Rosemont HospitalUniversity of Montreál Key Laboratory of Preclinical Study for New Drugs of Gansu Province Lanzhou University

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 characteri

关键词： Citraconic anhydride Inclusion bodies Recombinant modified C-reactive protein

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C-reactive protein in gallbladder diseases:diagnostic and therapeutic insights

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Biophysics Reports 2020年第Z1期6卷 49-67页

作者： Ibraheem M.Rajab Daniel Majerczyk Margaret E.Olson Jenna M.B.Addams Mihee L.Choe Matthew S.Nelson lawrence a.potempa College of Pharmacy Roosevelt University

The gallbladder is an important component of the hepatobiliary system whose primary function is to aid in digestion of foodstuffs, excretion of drugs and facilitate removal of waste products from the *** gall bladder is principally a storage organ for bile, chemically modified salts and acids of cholesterol which are synthesized in the liver and which function as surfactants to solubilize fatty substances of limited aqueous solubility. Any disruption in the amount or activity of bile surfactant can lead to an accumulation of insoluble molecular clumps(e.g., gallstones) that deposit in and obstruct fluid movement within the gallbladder, and which can lead to pathological congestion and tissue *** natural host defense response to any event that causes tissue damage is to stimulate inflammation,which non-specifically but aggressively reacts to the stimuli so to remove the cause and repair damaged tissues. The C-reactive protein(CRP) is a primarily hepatically produced serum protein whose blood levels increase within 6–10 h of any tissue-damaging event. The extent with which it increases correlates with the level of tissue damage and associated inflammation. CRP levels are reported to be of value in diagnosing acute cholecystitis severity, in predicting the outcome and prognosis of cancerassociated gallbladder resection, and in helping identify cystic structures during emergency laparoscopic cholecystectomies. As an understanding of distinctive CRP structural isoforms is evolving, its role not only as a biomarker but as regulator of both physiologic and pathophysiologic processes of inflammation may be relevant in the understanding of and treatment approaches for gallbladderassociated disease.

关键词： C-reactive protein Monomeric C-reactive Inflammation Gallbladder Gallstone Cholecystitis Cholesterol

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