MUTYH adenine DNA glycosylase and its homologous protein
(collectively
MutY) are typical DNA glycosylases with a [4Fe4S] cluster and a helix–hairpin–helix
(HhH) motif in its structure. In the present work, the binding behaviors
of the MutY protein to dsDNA containing different base mismatches
were investigated. The type and distribution of base mismatch in the
dsDNA chain were found to influence the DNA–protein binding
interaction greatly. The [4Fe4S] cluster of the MutY protein is able
to identify a G-A mismatch in the dsDNA chain specifically by monitoring
the anomalies of charge transport in the dsDNA chain, allowing the
entrance of the identified dsDNA chain into the internal cavity of
the MutY protein and the strong DNA–protein binding at the
HhH motif of the protein through multiple H-bonds. The dsDNA chain
with a centrally located G-A mismatch is thus functionalized on mesoporous
silica (MSN) via amination reaction, and the obtained dsDNA(G-A)@MSN
is used as a powerful sorbent for the selective capturing of the MutY
protein from complex samples. By using 0.5% NH3·H2O (m/v) as a stripping reagent, efficient isolation of the
MutY protein from different cell lines and bacteria is achieved and
the recovered MutY protein is demonstrated to maintain favorable DNA
adenine glycosylase activity.
Glutathione S-transferases (GSTs) are important type-II detoxification enzymes that protect DNA and proteins from damage and are often used as protein tags for the expression of fusion proteins. In the present work, octa-aminopropyl caged polyhedral oligomeric silsesquioxane (OA–POSS) was prepared via acid-catalyzed hydrolysis of 3-aminopropyltriethoxysilane and polymerized on the surface of graphene oxide (GO) through an amidation reaction. Glutathione (GSH) was then modified to GO–POSS through a Michael addition reaction to obtain a GSH-functionalized GO–POSS composite (GPG). The structure and characteristics of the as-prepared GPG composite were characterized using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), thermogravity analysis, and surface charge analysis. The specific binding interactions between glutathione and GST gave GPG favorable adsorption selectivity towards GST, and other proteins did not affect GST adsorption. The adsorption behavior of GST on the GPG composite conformed to the Langmuir isotherm model, and the adsorption capacity of GST was high up to 364.94 mg g−1 under optimal conditions. The GPG-based solid-phase adsorption process was applied to the extraction of GST from a crude enzyme solution of pig liver, and high-purity GST was obtained via SDS-PAGE identification.
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