Beldarraín A, Cruz Y, Cruz O, Navarro M, Gil M
Processes and Systems Evaluation Department, Production Plant, CIGB (Center for Genetic Engineering and Biotechnology), P.O. Box 6162, Havana, Cuba.
Biotechnol Appl Biochem. 2001 Jun;33(3):173-82. doi: 10.1042/ba20010001.
Recombinant human interferon alpha2b was expressed intracellularly in Escherichia coli as insoluble aggregates using a new expression vector, and was purified to homogeneity using essentially two-step chromatographic procedures, i.e. immobilized metal-ion-affinity chromatography and reversed-phase HPLC. The established purification process is highly reproducible and leads to a total recovery of approx. 12% with a specific biological activity of higher than 1x10(8) i.u./mg of protein, which is comparable with the international requirement for interferon alpha2b. For purified protein we report conformational stability as a function of pH and temperature using differential scanning calorimetry and CD. Thermal unfolding as a function of pH showed only one endotherm at a temperature higher than 45 degrees C, and was reversible at pH 2-3.75 and irreversible at pH 4-10. At pH 7.0, the most stable condition, the conformational stability depends on protein concentration and ionic strength. The highly helical secondary structure is very conserved over the whole pH range studied, including at high temperatures.
重组人干扰素α2b利用一种新的表达载体在大肠杆菌中进行胞内表达，形成不溶性聚集体，然后通过基本的两步色谱法，即固定化金属离子亲和色谱法和反相高效液相色谱法，纯化至同质。所建立的纯化工艺具有高度的可重复性，总回收率约为12%，比活性高于1×10⁸国际单位/毫克蛋白质，这与干扰素α2b的国际要求相当。对于纯化后的蛋白质，我们使用差示扫描量热法和圆二色光谱法报告了其构象稳定性随pH值和温度的变化情况。热变性随pH值的变化表明，在高于45℃的温度下只有一个吸热峰，在pH 2 - 3.75时是可逆的，在pH 4 - 10时是不可逆的。在pH 7.0（最稳定的条件）下，构象稳定性取决于蛋白质浓度和离子强度。在所研究的整个pH范围内，包括在高温下，高度螺旋的二级结构都非常保守。
