His-tag的人胞浆磷脂酶A_2的C2结构域的表达、纯化及性质

Expression, Purification and Characterization of the His-tag C2 Domain of Human Cytosolic Phospholipase A_2

带有His tag的人胞浆磷脂酶A2 的C2结构域高效表达 ,用内源荧光的变化测定了其稳定性和其与钙离子结合的结合常数 .结果表明 ,带有His tag的C2结构域仍可有效用于研究其折叠及其与钙离子的协同性结合 ,温度从 2 2℃升高到 35℃时 ,C2结构域和钙离子结合的协同性程度显著增强 .

The high expression of the His tagged C2 domain of human cytosolic phospholipase A 2, its stability and calcium association constants were reported. The results showed that the His tagged C2 domain could be efficiently used to investigate its folding stability with the Gibbs free energy of 15 67 kJ·mol -1 and the m value of 4 83 kJ·mol -2 ·L at pH 7 0, using urea as a denaturant, and to measure its calcium binding constant for each calcium binding site as (1 89±0 18)×10 5 mol -1 ·L and (6 06±0 59)×10 5 mol -1 ·L at 22℃, and (2 3±0 19)×10 4 mol -1 ·L and (3 1±0 24)×10 5 mol -1 ·L at 35℃, respectively, via employing the two binding sites formula. Raising temperature could significantly enhance the calcium binding cooperativity.