2012
DOI: 10.1107/s1744309112011025
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Purification, crystallization and preliminary X-ray crystallographic analysis of 3-ketosteroid Δ1-dehydrogenase fromRhodococcus erythropolisSQ1

Abstract: 3-Ketosteroid Á 1 -dehydrogenase plays a crucial role in the early steps of steroid degradation by introducing a double bond between the C1 and C2 atoms of the A-ring of its 3-ketosteroid substrates. The 3-ketosteroid Á 1 -dehydrogenase from Rhodococcus erythropolis SQ1, a 56 kDa flavoprotein, was crystallized using the sitting-drop vapour-diffusion method at room temperature. The crystals grew in various buffers over a wide pH range (from pH 5.5 to 10.5), but the best crystallization condition consisted of 2%… Show more

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Cited by 18 publications
(21 citation statements)
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“…Protein Expression and Purification-A protocol for ⌬ 1 -KSTD1 expression has been established (2), as well as a procedure for its purification (17). In brief, Escherichia coli strain BL21(DE3) cells harboring the pET15b-kstD1 plasmid (2) were grown at 290 K. N-terminally His-tagged ⌬ 1 -KSTD1 was overexpressed by induction with 100 M isopropyl-␤-D-1-thiogalactopyranoside for 48 h. After cell lysis, ⌬ 1 -KSTD1 was purified by immobilized Ni 2ϩ affinity chromatography (HisTrap HP; GE Healthcare), followed by anion exchange chromatography (Resource Q; GE Healthcare) and size exclusion chromatography (Superdex 200 10/300 GL; GE Healthcare).…”
Section: Methodsmentioning
confidence: 99%
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“…Protein Expression and Purification-A protocol for ⌬ 1 -KSTD1 expression has been established (2), as well as a procedure for its purification (17). In brief, Escherichia coli strain BL21(DE3) cells harboring the pET15b-kstD1 plasmid (2) were grown at 290 K. N-terminally His-tagged ⌬ 1 -KSTD1 was overexpressed by induction with 100 M isopropyl-␤-D-1-thiogalactopyranoside for 48 h. After cell lysis, ⌬ 1 -KSTD1 was purified by immobilized Ni 2ϩ affinity chromatography (HisTrap HP; GE Healthcare), followed by anion exchange chromatography (Resource Q; GE Healthcare) and size exclusion chromatography (Superdex 200 10/300 GL; GE Healthcare).…”
Section: Methodsmentioning
confidence: 99%
“…Protein Crystallization-⌬ 1 -KSTD1 was crystallized using the sitting drop, vapor diffusion method at room temperature (17). Typically, 0.30-l drops comprising 0.15 l of protein sample (15 mg ml Ϫ1 protein) mixed with 0.15 l of reservoir solution (2% (v/v) polyethylene glycol 400, 0.1 M HEPES were equilibrated against wells containing 50 l of the reservoir solution.…”
Section: Methodsmentioning
confidence: 99%
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