A bottleneck
in fragment-based lead development is the lack of
systematic approaches to elaborate the initial fragment hits, which
usually bind with low affinity to their target. Herein, we describe
an analysis using X-ray crystallography of a diverse library of compounds
prepared using microscale parallel synthesis. This approach yielded
an 8-fold increase in affinity and detailed structural information
for the resulting complex, providing an efficient and broadly applicable
approach to early fragment development.
A predicted difference in SF ferritin concentrations in patients with knee OA was confirmed. Concentrations of ferritin in the SF were found to be two- to threefold higher in knee OA patients with HFE gene mutations compared to wt patients. This finding is consistent with the possibility that, in OA patients with HFE gene mutations, localized iron overload may contribute either directly or indirectly to osteochondral damage, possibly in a similar way to that which occurs in the arthropathy that complicates HH.
Adenovirus protein VII is a highly cationic core protein that forms a nucleosome-like structure in the adenovirus core by condensing DNA in combination with protein V and mu. It has been proposed that protein VII could condense DNA in a manner analogous to mammalian histones. Due to the lack of an expression and purification protocol, the interactions between protein VII and DNA are poorly understood. In this study we describe methods for the purification of biologically active recombinant protein VII using an E. coli expression system. We expressed a cleavable fusion of protein VII with thioredoxin and established methods for purification of this fusion protein in denatured form. We describe an efficient method for resolving the cleavage products to obtain pure protein VII using hydroxyapatite column chromatography. Mass spectroscopy data confirmed its mass and purity to be 19.4 kDa and >98 %, respectively. Purified recombinant protein VII spontaneously condensed dsDNA to form particles, as shown by dye exclusion assay, electrophoretic mobility shift assay and nuclease protection assay. Additionally, an in vitro bioluminescence assay revealed that protein VII can be used to enhance the transfection of mammalian cells with lipofectamine/DNA complexes. The availability of recombinant protein VII will facilitate future studies of the structure of the adenovirus core. Improved understanding of the structure and function of protein VII will be valuable in elucidating the mechanism of adenoviral DNA condensation, defining the morphology of the adenovirus core and establishing the mechanism by which adenoviral DNA enters the nucleus.
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