Expression, purification, crystallization and preliminary diffraction data characterization of Escherichia coli ribonuclease II (RNase II)
- Division of Biological Chemistry, ITQB - Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Apt. 127, 2781-901 Oeiras (Portugal)
- Division of Biology, ITQB - Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Apt. 127, 2781-901 Oeiras (Portugal)
Diffraction data from E. coli RNase II crystals of wild type and of an inactive mutant and its SeMet-derivative form were obtained to 2.44 and 2.74 Å resolution, providing a set of preliminary phases. An improved purification protocol allowed higher reproducibility in the crystallization of the mutant form. RNA degradation is important in the post-transcriptional control of gene expression. The processing, degradation and quality control of RNA is performed by many different classes of ribonucleases. Ribonuclease II (RNase II) is a 643-amino-acid enzyme that degrades single-stranded RNA from its 3′-end, releasing ribonucleoside 5′-monophosphates. RNase II was expressed both as the wild type and as a D209N mutant form. The latter was also produced as an SeMet derivative. The various protein forms were crystallized using the vapour-diffusion method. Wild-type RNase II was crystallized in two crystal forms, both of which belonged to space group P2{sub 1}. X-ray diffraction data were collected to 2.44 and 2.75 Å resolution, with unit-cell parameters a = 56.8, b = 125.7, c = 66.2 Å, β = 111.9° and a = 119.6, b = 57.2, c = 121.2 Å, β = 99.7°, respectively. The RNase II D209N mutant gave crystals that belonged to space group P6{sub 5}, with unit-cell parameters a = b = 86.3, c = 279.2 Å, and diffracted to 2.74 Å. Diffraction data from the mutant and its SeMet derivative enabled the determination of a partial Se-atom substructure by SIRAS.
- OSTI ID:
- 22360203
- Journal Information:
- Acta Crystallographica. Section F, Vol. 62, Issue Pt 7; Other Information: PMCID: PMC2242952; PMID: 16820694; PUBLISHER-ID: gj5004; OAI: oai:pubmedcentral.nih.gov:2242952; Copyright (c) International Union of Crystallography 2006; Country of input: International Atomic Energy Agency (IAEA); ISSN 1744-3091
- Country of Publication:
- United Kingdom
- Language:
- English
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