Purification of recombinant C-reactive protein mutants

Thirumalai, Avinash; Singh, Sanjay K.; Hammond, David J.; Gang, Toh B.; Ngwa, Donald N.; Pathak, Asmita; Agrawal, Alok

doi:10.1016/j.jim.2017.01.011

Cited by 8 publications

(5 citation statements)

References 67 publications

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“…Eluted E-CRP-1 was immediately dialyzed against TBS containing 2 mM CaCl 2 , stored at 4 • C, and was used within a week. WT CRP and all other CRP mutants including E-CRP-2 were purified as described previously (49). The purity of CRP preparations was confirmed by denaturing 4-20% SDS-PAGE under reducing conditions.…”

Section: Expression and Purification Of Crpmentioning

confidence: 99%

Treatment of Pneumococcal Infection by Using Engineered Human C-Reactive Protein in a Mouse Model

et al. 2020

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C-reactive protein (CRP) binds to several species of bacterial pathogens including Streptococcus pneumoniae. Experiments in mice have revealed that one of the functions of CRP is to protect against pneumococcal infection by binding to pneumococci and activating the complement system. For protection, however, CRP must be injected into mice within a few hours of administering pneumococci, that is, CRP is protective against early-stage infection but not against late-stage infection. It is assumed that CRP cannot protect if pneumococci got time to recruit complement inhibitor factor H on their surface to become complement attack-resistant. Since the conformation of CRP is altered under inflammatory conditions and altered CRP binds to immobilized factor H also, we hypothesized that in order to protect against latestage infection, CRP needed to change its structure and that was not happening in mice. Accordingly, we engineered CRP molecules (E-CRP) which bind to factor H on pneumococci but do not bind to factor H on any host cell in the blood. We found that E-CRP, in cooperation with wild-type CRP, was protective regardless of the timing of administering E-CRP into mice. We conclude that CRP acts via two different conformations to execute its anti-pneumococcal function and a model for the mechanism of action of CRP is proposed. These results suggest that pre-modified CRP, such as E-CRP, is therapeutically beneficial to decrease bacteremia in pneumococcal infection. Our findings may also have implications for infections with antibiotic-resistant pneumococcal strains and for infections with other bacterial species that use host proteins to evade complement-mediated killing.

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Section: Expression and Purification Of Crpmentioning

confidence: 99%

Treatment of Pneumococcal Infection by Using Engineered Human C-Reactive Protein in a Mouse Model

et al. 2020

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“…Upon adding TUA2 or ATS, CRP was converted to hyper-acidified fusion forms (TUA2-CRP, ATS-CRP) with much more negative charges (Supplementary Table 1), and likely become difficult to interact for aggregation due to strengthened electrostatic repulsion between similarly charged fusion moieties, thus allowing sufficient time for correct folding to achieve an elevated solubility 63,67 . Prospectively, this strategy could be utilized for scale preparation of soluble CRP to meet its diverse uses, alternative to the previous approaches 27,41,68–70 . Meanwhile, hyper-acidic minipeptides TUA2, ATS might also take a great deal of application potential in protein engineering and further genetic engineering.…”

Section: Discussionmentioning

confidence: 99%

Hyper-acidic fusion minipeptides escort the intrinsic antioxidative ability of the pattern recognition receptor CRP in non-animal organisms

Zhang

Liu

et al. 2019

Sci Rep

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C-reactive protein (CRP) is widely used as a biomarker of inflammation. It plays important roles in innate immunity response as a member of pattern recognition receptors, by binding oxidation-specific epitopes including some intermediates of lipid oxidative chain reaction. The inferred antioxidative ability of CRP was ever demonstrated by only few in vitro evidences, and needs to be clarified especially in vivo . Herein, we expressed human CRP in three representative non-animal organisms ( Escherichia coli , Saccharomyces cerevisiae , and tobacco) inherently lacking the milieu for CRP signalling, and found CRP did possess an intrinsic antioxidative ability. Heterologous CRP could confer increased oxidative resistance in its recombinant E . coli and yeast cells and transgenic tobaccos. We also revealed a positive correlation between the antioxidative effect of CRP and its solubility. Only soluble CRP could exhibit distinct antioxidative activity, while the CRP aggregates might be instead toxic (probably pro-oxidative) to cells. Moreover, fusion with hyper-acidic minipeptides could remarkably improve CRP solubility, and meanwhile guarantee or enhance CRP antioxidative ability. These results not only provide a new insight for understanding the etiology of CRP-involved inflammations and diseases, and also endorse a potential of CRP biotechnological applications in developing new pharmaceutical therapies and improving plant oxidative resistance.

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“…Native WT CRP was purified from discarded human pleural fluid as described previously ( 43 ). Recombinant mutant CRP Y40F/E42Q was expressed in CHO cells using the ExpiCHO Expression System (Thermo Fisher Scientific) and purified from the culture supernatant exactly as described for WT CRP ( 16 ).…”

Section: Methodsmentioning

confidence: 99%

C-reactive protein lowers the serum level of IL-17, but not TNF-α, and decreases the incidence of collagen-induced arthritis in mice

Singh,

Prislovsky,

Ngwa

et al. 2024

Front. Immunol.

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The biosynthesis of C-reactive protein (CRP) in the liver is increased in inflammatory diseases including rheumatoid arthritis. Previously published data suggest a protective function of CRP in arthritis; however, the mechanism of action of CRP remains undefined. The aim of this study was to evaluate the effects of human CRP on the development of collagen-induced arthritis (CIA) in mice which is an animal model of autoimmune inflammatory arthritis. Two CRP species were employed: wild-type CRP which binds to aggregated IgG at acidic pH and a CRP mutant which binds to aggregated IgG at physiological pH. Ten CRP injections were given on alternate days during the development of CIA. Both wild-type and mutant CRP reduced the incidence of CIA, that is, reduced the number of mice developing CIA; however, CRP did not affect the severity of the disease in arthritic mice. The serum levels of IL-17, IL-6, TNF-α, IL-10, IL-2 and IL-1β were measured: both wild-type and mutant CRP decreased the level of IL-17 and IL-6 but not of TNF-α, IL-10, IL-2 and IL-1β. These data suggest that CRP recognizes and binds to immune complexes, although it was not clear whether CRP functioned in its native pentameric or in its structurally altered pentameric form in the CIA model. Consequently, ligand-complexed CRP, through an as-yet undefined mechanism, directly or indirectly, inhibits the production of IL-17 and eventually protects against the initiation of the development of arthritis. The data also suggest that IL-17, not TNF-α, is critical for the development of autoimmune inflammatory arthritis.

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Purification of recombinant C-reactive protein mutants

Cited by 8 publications

References 67 publications

Treatment of Pneumococcal Infection by Using Engineered Human C-Reactive Protein in a Mouse Model

Treatment of Pneumococcal Infection by Using Engineered Human C-Reactive Protein in a Mouse Model

Hyper-acidic fusion minipeptides escort the intrinsic antioxidative ability of the pattern recognition receptor CRP in non-animal organisms

C-reactive protein lowers the serum level of IL-17, but not TNF-α, and decreases the incidence of collagen-induced arthritis in mice

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