Purification, crystallization, X-ray diffraction analysis and phasing of an engineered single-chain PvuII restriction endonuclease
- Department of Science, Agricultural University of Athens, Athens (Greece)
- Institute of Molecular Biology and Biotechnology (IMBB), PO Box 1527, GR-71110 Heraklion, Crete (Greece)
- Department of Agricultural Biotechnology, Agricultural University of Athens, Athens (Greece)
- Department of Biology, University of Crete, PO Box 2208, GR-71110 Heraklion, Crete (Greece)
PvuII is the first type II restriction endonuclease to be converted from its wild-type homodimeric form into an enzymatically active single-chain variant. The enzyme was crystallized and phasing was successfully performed by molecular replacement. The restriction endonuclease PvuII from Proteus vulgaris has been converted from its wild-type homodimeric form into the enzymatically active single-chain variant scPvuII by tandemly joining the two subunits through the peptide linker Gly-Ser-Gly-Gly. scPvuII, which is suitable for the development of programmed restriction endonucleases for highly specific DNA cleavage, was purified and crystallized. The crystals diffract to a resolution of 2.35 Å and belong to space group P4{sub 2}, with unit-cell parameters a = b = 101.92, c = 100.28 Å and two molecules per asymmetric unit. Phasing was successfully performed by molecular replacement.
- OSTI ID:
- 22360397
- Journal Information:
- Acta Crystallographica. Section F, Vol. 63, Issue Pt 10; Other Information: PMCID: PMC2339719; PMID: 17909283; PUBLISHER-ID: bw5199; OAI: oai:pubmedcentral.nih.gov:2339719; Copyright (c) International Union of Crystallography 2007; Country of input: International Atomic Energy Agency (IAEA); ISSN 1744-3091
- Country of Publication:
- United Kingdom
- Language:
- English
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