鼠脑驱动蛋白的表达、纯化和结晶

Expression, purification and crystallization of rat brain kinesin

摘要:

驱动蛋白是一类利用水解ATP为ADP和磷酸的过程中释放的能量沿微管系统运动的蛋白。为了研究ATP中储存的化学能是如何转化为驱动蛋白的机械动能，鼠脑驱动蛋白的相关N-端区域在BL21-Codon Plus(DE3)-RP感受态大肠杆菌细胞中大量地表达。通过SP-强阳离子交换色谱和分子筛色谱的两步骤纯化，蛋白最终产量高达10 mg/L细胞培养液，蛋白纯度可以达到95%以上。纯化的蛋白具有水解ATP酶的活力，并与驱动蛋白抗体有特异性的反应。驱动蛋白可以在如下条件结晶：1.7 mol/L (NH4)2SO4, 500 mmol/L NaCl, 20% glycerol。晶体衍射的分辨率可以达到2.0 ?。

Abstract:

Kinesin is a motor protein that uses the energy from ATP hydrolysis to move along the microtubule system. To investigate how the chemical energy stored in ATP is converted to mechanical movement, the corresponding N-terminal region of rat brain kinesin was expressed in BL21-Codon Plus (DE3)-RP competent cells. After SP-cation exchange chromatography and size exclusion chromatography, the protein yield reached 10 mg/L culture with the purity above 95%. The purified protein had ATPase activity and specifically reacted with the kinesin antibody in the Western blotting analysis. The purified kinesin was crystallized under the following condition: 1.7 mol/L (NH4)2SO4, 500 mmol/L NaCl, 20% glycerol. The kinesin crystal can diffract up to 2.0 ? resolution.