Cloning, expression, purification, crystallization and initial crystallographic analysis of the lysine-biosynthesis LysX protein from Thermus thermophilus HB8


Marina N. Vassylyevaa,b, Hiroaki Sakaia, Takanori Matsuuraa, Shun-ichi Sekineb,c, Makoto Nishiyamad, Takaho Teradaa,b, Mikako Shirouzua,b, Seiki Kuramitsua,b,e, Dmitry G. Vassylyevb, c, and Shigeyuki Yokoyamaa,b,c,f

aGenomic Sciences Center, RIKEN Yokohama Institute, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan,
bStructurome Project, RIKEN Harima Institute at SPring-8, 1-1-1 Kouto, Mikazuki-cho, Sayo, Hyogo 679-5148, Japan,
cCellular Signaling Laboratory, RIKEN Harima Institute at SPring-8, 1-1-1 Kouto, Mikazuki-cho, Sayo, Hyogo 679-5148, Japan,
dBiotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan,
eDepartment of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan,
fDepartment of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Bunkyo-Ku, Tokyo 113-0033, Japan

Acta Crystallographica D59(9), 1651-1652 (2003)

The gene encoding LysX, an essential component of the lysine-biosynthesis pathway in Thermus thermophilus (molecular weight ≃ 31 000 Da), was cloned and expressed and the purified protein was crystallized by the hanging-drop vapour-diffusion technique in two different space groups, C2 (unit-cell parameters a = 124.7, b = 51.4, c = 103.6 Å, β = 122.8°) and R3 (a = b = 122.6, c = 97.6 Å). Crystals improved by macroseeding diffracted to beyond 2.3 and 3 Å resolution for the C2 and R3 crystal forms, respectively. Complete diffraction data sets were collected for the C2 and R3 crystal forms at 2.5 and 3.1 Å resolution, respectively. Crystals of selenomethionine-containing LysX protein were obtained by cross-microseeding, using the native microcrystals as a seed. Structure determination is now in progress.




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MATSUURA Takanori
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