Purification, crystallization and preliminary X-ray diffraction of SecDF, a translocon-associated membrane protein, from Thermus thermophilus.
Thermus thermophilus has a multi-path membrane protein, TSecDF, as a single-chain homologue of Escherichia coli SecD and SecF, which form a translocon-associated complex required for efficient preprotein translocation and membrane-protein integration. Here, the cloning, expression in E. coli, purification and crystallization of TSecDF are reported. Overproduced TSecDF was solubilized with dodecylmaltoside, chromatographically purified and crystallized by vapour diffusion in the presence of polyethylene glycol. The crystals yielded a maximum resolution of 4.2 angstroms upon X-ray irradiation, revealing that they belonged to space group P4(3)2(1)2. Attempts were made to improve the diffraction quality of the crystals by combinations of micro-stirring, laser-light irradiation and dehydration, which led to the eventual collection of complete data sets at 3.74 angstroms resolution and preliminary success in the single-wavelength anomalous dispersion analysis. These results provide information that is essential for the determination of the three-dimensional structure of this important membrane component of the protein-translocation machinery.
Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan.
Tomoya
T
Tsukazaki
Hiroyuki
H
Mori
Shuya
S
Fukai
Tomoyuki
T
Numata
Anna
A
Perederina
Hiroaki
H
Adachi
Hiroyoshi
H
Matsumura
Kazufumi
K
Takano
Satoshi
S
Murakami
Tsuyoshi
T
Inoue
Yusuke
Y
Mori
Takatomo
T
Sasaki
Dmitry G
DG
Vassylyev
Osamu
O
Nureki
Koreaki
K
Ito
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D003460:Crystallization; D017931:DNA Primers; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D021381:Protein Transport; D016963:Thermus thermophilus / Q000737:chemistry*; D014961:X-Ray Diffraction
D001426:Bacterial Proteins; D017931:DNA Primers; D008565:Membrane Proteins