ABSTRACT
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.
Subject(s)
Deoxyribonucleases, Type II Site-Specific/chemistry , Protein Engineering , Crystallization , Deoxyribonucleases, Type II Site-Specific/genetics , Deoxyribonucleases, Type II Site-Specific/isolation & purification , Escherichia coli/enzymology , Escherichia coli/genetics , Protein Engineering/methods , Proteus vulgaris/enzymology , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , X-Ray DiffractionABSTRACT
The Bacillus cereus BC1534 protein, a putative deacetylase from the LmbE family, has been purified to homogeneity and crystallized using the hanging-drop vapour-diffusion method. Crystals of the 26 kDa protein grown from MPD and acetate buffer belong to space group R32, with unit-cell parameters a = b = 76.7, c = 410.5 A (in the hexagonal setting). A complete native data set was collected to a resolution of 2.5 A from a single cryoprotected crystal using synchrotron radiation. As BC1534 shows significant sequence homology with an LmbE-like protein of known structure from Thermus thermophilus, molecular replacement will be used for crystal structure determination.
