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Solubilization and purification of recombinant modified C-reactive protein from
inclusion bodies using reversible anhydride modification
#MMPMID26942216
Potempa LA
; Yao ZY
; Ji SR
; Filep JG
; Wu Y
Biophys Rep
2015[]; 1
(?): 18-33
PMID26942216
show ga
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., mCRP), that is expressed when CRP subunits are dissociated into monomeric
structures. The free mCRP 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.,
pCRP). As specific reagents have been developed to identify and quantify mCRP, it
has become apparent that this isoform can be formed spontaneously in calcium-free
solutions. Furthermore, mCRP can be expressed on perturbed cell membranes with as
little as 24-48 h incubation in tissue culture. Because mCRP has the same size as
pCRP subunits as evaluated by SDS-PAGE, its presence in a pCRP 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 mCRP, 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
mCRP rather than on pCRP. To provide a reliable, abundant supply of mCRP for
separate and comparable studies, a recombinant protein was engineered and
expressed in E. coli (i.e., recombinant mCRP or r(m)CRP). 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 r(m)CRP and allow for chromatographic
purification in high yield and free of contaminating endotoxin. Furthermore, the
purified r(m)CRP reagent represents an excellent comparable protein to the
biologically produced mCRP and as a distinctive reagent from pCRP. 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.
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