Overproduction, purification, crystallization and preliminary X-ray diffraction analysis of Trypanosoma brucei gambiense glycerol kinase
Overproduction, purification, crystallization and preliminary X-ray diffraction analysis of Trypanosoma brucei gambiense glycerol kinase
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DOI：
10.1107/s1744309110000369
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发表时间：
2010-03-01
影响因子：
0.9
通讯作者：
Kita, Kiyoshi
中科院分区：
文献类型：
--
作者：
Balogun, Emmanuel Oluwadare;Inaoka, Daniel Ken;Kita, Kiyoshi
在人类锥虫的血流形式中，甘油激酶（GK; EC 2.7.1.30）是能量代谢所必需的九种糖体区室化酶之一。本研究采用聚乙二醇400沉淀法，过量表达并纯化了N端带有His（6）标签的重组布氏冈比亚锥虫GK（rTbgGK）。2.75埃分辨率的完整X射线衍射数据表明，该晶体属于斜方晶系空间群P2（1）2（1）2（1），晶胞参数a = 63.84，b = 121.50，B = 154.59埃。在不对称单元中存在两个rTbgGK分子给出了2.5埃（3）Da（-1）的马修斯系数（V-M），对应于50%的溶剂含量。
In the bloodstream forms of human trypanosomes, glycerol kinase (GK; EC 2.7.1.30) is one of the nine glycosomally compartmentalized enzymes that are essential for energy metabolism. In this study, a recombinant Trypanosoma brucei gambiense GK (rTbgGK) with an N-terminal cleavable His(6) tag was overexpressed, purified to homogeneity and crystallized by the sitting-drop vapour-diffusion method using PEG 400 as a precipitant. A complete X-ray diffraction data set to 2.75 angstrom resolution indicated that the crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 63.84, b = 121.50, c = 154.59 angstrom. The presence of two rTbgGK molecules in the asymmetric unit gives a Matthews coefficient (V-M) of 2.5 angstrom(3) Da(-1), corresponding to 50% solvent content.