pHluorin-assisted expression, purification, crystallization and X-ray diffraction data analysis of the C-terminal domain of the HsdR subunit of the Escherichia coli type I restriction-modification system EcoR124I
Language English Country United States Media print-electronic
Document type Journal Article
PubMed
27599856
PubMed Central
PMC5012205
DOI
10.1107/s2053230x16011626
PII: S2053230X16011626
Knihovny.cz E-resources
- Keywords
- EcoR124I, Escherichia coli, GFP, HsdR, pHluorin, restriction-modification system,
- MeSH
- X-Ray Diffraction MeSH
- Escherichia coli chemistry enzymology genetics MeSH
- Gene Expression MeSH
- Cloning, Molecular MeSH
- Crystallization MeSH
- Crystallography, X-Ray MeSH
- Plasmids chemistry metabolism MeSH
- Protein Subunits chemistry genetics metabolism MeSH
- Escherichia coli Proteins chemistry genetics metabolism MeSH
- Recombinant Fusion Proteins chemistry genetics metabolism MeSH
- Deoxyribonucleases, Type I Site-Specific chemistry genetics metabolism MeSH
- Amino Acid Sequence MeSH
- Green Fluorescent Proteins chemistry genetics metabolism MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- endodeoxyribonuclease EcoR124I MeSH Browser
- HsdR protein, E coli MeSH Browser
- PHluorin MeSH Browser
- Protein Subunits MeSH
- Escherichia coli Proteins MeSH
- Recombinant Fusion Proteins MeSH
- Deoxyribonucleases, Type I Site-Specific MeSH
- Green Fluorescent Proteins MeSH
The HsdR subunit of the type I restriction-modification system EcoR124I is responsible for the translocation as well as the restriction activity of the whole complex consisting of the HsdR, HsdM and HsdS subunits, and while crystal structures are available for the wild type and several mutants, the C-terminal domain comprising approximately 150 residues was not resolved in any of these structures. Here, three fusion constructs with the GFP variant pHluorin developed to overexpress, purify and crystallize the C-terminal domain of HsdR are reported. The shortest of the three encompassed HsdR residues 887-1038 and yielded crystals that belonged to the orthorhombic space group C2221, with unit-cell parameters a = 83.42, b = 176.58, c = 126.03 Å, α = β = γ = 90.00° and two molecules in the asymmetric unit (VM = 2.55 Å(3) Da(-1), solvent content 50.47%). X-ray diffraction data were collected to a resolution of 2.45 Å.
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