Purification, crystallization, X-ray diffraction analysis and phasing of an engineered single-chain PvuII restriction endonuclease.
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 A and belong to space group P4(2), with unit-cell parameters a = b = 101.92, c = 100.28 A and two molecules per asymmetric unit. Phasing was successfully performed by molecular replacement.
Meramveliotaki
Chrysi
C
Department of Science, Agricultural University of Athens, Athens, Greece.
Kotsifaki
Dina
D
Androulaki
Maria
M
Hountas
Athanasios
A
Eliopoulos
Elias
E
Kokkinidis
Michael
M
D003460:Crystallization; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015202:Protein Engineering / Q000379:methods; D011514:Proteus vulgaris / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014961:X-Ray Diffraction
D011994:Recombinant Proteins; C025630:CAGCTG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific