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Heterologously Expressed Polypeptide from the Yeast Meiotic Gene HOP1 Binds Preferentially to Yeast DNA

AuthorsAlché Ramírez, Juan de Dios; Paul, Elizabeth; Dickinson, Hugh
Fusion protein
Biotinylated tag
Gel-shift assay
Issue DateJul-1999
CitationProtein Expression and Purification 16(2): 251-260 (1999)
AbstractHOP1 protein, present in sporulating cells of Saccharomyces cerevisiae and believed to be a component of the synaptonemal complex, has been expressed in Escherichia coli fused to a biotinylated tag protein. Once solubilized from bacterial inclusion bodies, the HOP1 fusion protein was purified by using a combination of avidin-affinity chromatography and gel filtration FPLC and refolded. Sequence comparisons indicate that the HOP1 gene product contains a zinc finger motif, which may confer DNA binding properties, and the recombinant polypeptide was used to assess the putative DNA binding properties of the product of native HOP1 protein using a gel-shift assay. Protein and protein–DNA complexes were detected by exploiting the affinity of streptavidin-alkaline phosphatase for the biotinylated tag protein after Western blotting. The HOP1 fusion protein bound unambiguously to digested genomic yeast DNA. This binding possessed some degree of specificity, was maintained under a wide range of salt concentrations, and was unaffected by the presence of high concentrations of competitor DNA (synthetic poly[dI-dC].poly[dI-dC]). In contrast, no shift was detected when the fusion protein was incubated with digested genomic DNA from Arabidopsis, or with λ/HindIII DNA. Incubation with digested genomic DNA from Lilium produced a small change in the mobility of the protein. The biotinylated tag protein failed to show any DNA binding activity. Scatchard analysis indicated an apparent yeast genomic DNA:HOP1 fusion protein dissociation constant of Kd = 5 × 10−7 M.
Description10 páginas, 8 figuras.
Publisher version (URL)http://dx.doi.org/10.1006/prep.1999.1052
Appears in Collections:(EEZ) Artículos
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