Expression, crystallization and preliminary diffraction studies of the Pseudomonas putida cytochrome P450cam operon repressor CamR
- Structural Biology Center, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540 (Japan)
- Department of Molecular Biology, Daiichi College of Pharmaceutical Sciences, Minami-ku, Fukuoka 815-8511 (Japan)
The P. putida cytochrome P450cam operon repressor CamR has been expressed in E. coli and crystallized in space group P2{sub 1}2{sub 1}2. The Pseudomonas putida cam repressor (CamR) is a homodimeric protein that binds to the camO DNA operator to inhibit the transcription of the cytochrome P450cam operon camDCAB. CamR has two functional domains: a regulatory domain and a DNA-binding domain. The binding of the inducer d-camphor to the regulatory domain renders the DNA-binding domain unable to bind camO. Native CamR and its selenomethionyl derivative have been overproduced in Escherichia coli and purified. Native CamR was crystallized under the following conditions: (i) 12–14% PEG 4000, 50 mM Na PIPES, 0.1 M KCl, 1% glycerol pH 7.3 at 288 K with and without camphor and (ii) 1.6 M P{sub i}, 50 mM Na PIPES, 2 mM camphor pH 6.7 at 278 K. The selenomethionyl derivative CamR did not crystallize under either of these conditions, but did crystallize using 12.5% PEG MME 550, 25 mM Na PIPES, 2.5 mM MgCl{sub 2} pH 7.3 at 298 K. Preliminary X-ray diffraction studies revealed the space group to be orthorhombic (P2{sub 1}2{sub 1}2), with unit-cell parameters a = 48.0, b = 73.3, c = 105.7 Å. Native and selenomethionyl derivative data sets were collected to 3 Å resolution at SPring-8 and the Photon Factory.
- OSTI ID:
- 22356052
- Journal Information:
- Acta Crystallographica. Section F, Vol. 61, Issue Pt 8; Other Information: PMCID: PMC1952358; PMID: 16511161; PUBLISHER-ID: ll5027; OAI: oai:pubmedcentral.nih.gov:1952358; Copyright (c) International Union of Crystallography 2005; Country of input: International Atomic Energy Agency (IAEA); ISSN 1744-3091
- Country of Publication:
- United Kingdom
- Language:
- English
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