TY - JOUR
T1 - Crystallization of the Bacillus subtilis SPP1 bacteriophage helicase loader protein G39P
AU - Bailey, S.
AU - Sedelnikova, S. E.
AU - Mesa, P.
AU - Ayora, S.
AU - Alonso, J. C.
AU - Rafferty, J. B.
PY - 2003/6/1
Y1 - 2003/6/1
N2 - The essential helicase loader protein G39P encoded by Bacillus subtilis SPP1 phage has been overproduced in Escherichia coli and purified. The wild-type protein has been crystallized by the hanging-drop vapour-diffusion method in a primitive hexagonal space group, probably P6122/P6522, but the crystals diffract to only 3.4 Å and are poorly reproducible. Mass-spectrometric analysis has revealed marked proteolytic cleavage from the C-terminus and the presence of a major species corresponding to deletion of the 14 C-terminal residues. Thus, a new variant of the protein (G39P112) has been engineered that corresponds to a 14-residue C-terminal truncation. The G39P112 variant has also been crystallized but now in a primitive orthorhombic form, probably P21212 or P212121, with unit-cell parameters a = 85.6, b = 89.7, c = 47.6 Å, with diffraction to 2.4 Å on a synchrotron source and with greatly improved reproducibility. Calculation of VM values for this G39P112 variant suggests the presence of three monomers in the asymmetric unit, corresponding to a solvent content of about 47%. A selenomethionine-incorporated form of the protein has been produced and a full three-wavelength MAD data collection undertaken.
AB - The essential helicase loader protein G39P encoded by Bacillus subtilis SPP1 phage has been overproduced in Escherichia coli and purified. The wild-type protein has been crystallized by the hanging-drop vapour-diffusion method in a primitive hexagonal space group, probably P6122/P6522, but the crystals diffract to only 3.4 Å and are poorly reproducible. Mass-spectrometric analysis has revealed marked proteolytic cleavage from the C-terminus and the presence of a major species corresponding to deletion of the 14 C-terminal residues. Thus, a new variant of the protein (G39P112) has been engineered that corresponds to a 14-residue C-terminal truncation. The G39P112 variant has also been crystallized but now in a primitive orthorhombic form, probably P21212 or P212121, with unit-cell parameters a = 85.6, b = 89.7, c = 47.6 Å, with diffraction to 2.4 Å on a synchrotron source and with greatly improved reproducibility. Calculation of VM values for this G39P112 variant suggests the presence of three monomers in the asymmetric unit, corresponding to a solvent content of about 47%. A selenomethionine-incorporated form of the protein has been produced and a full three-wavelength MAD data collection undertaken.
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U2 - 10.1107/S0907444903007236
DO - 10.1107/S0907444903007236
M3 - Article
C2 - 12777784
AN - SCOPUS:0038721267
SN - 0907-4449
VL - 59
SP - 1090
EP - 1092
JO - Acta Crystallographica - Section D Biological Crystallography
JF - Acta Crystallographica - Section D Biological Crystallography
IS - 6
ER -