基于超滤膜电渗流动建立尿素梯度及其应用于蛋白质复性

Generation of Urea Gradient by Electroosmosis Through Ultrafiltration Membrane and Its Application to Protein Refolding

建立了由超滤膜隔开的5腔室电泳装置,利用超滤膜的电渗流动来建立中间腔室中沿流动方向上的尿素梯度,在此梯度中实现蛋白质与尿素及其它变性剂的分离,并进而导致蛋白质的复性. 建立了描述尿素梯度的模型并进行了实验验证,考察了牛血清白蛋白在此尿素梯度中的复性,证实了此技术的可行性并显示了很好的应用前景.

An ultrafiltration membrane-spaced 5-compartment electrolyzer was developed for protein refolding conducted in a longitudinal concentration gradient of urea in the central compartment, as a results of urea transport between central and elution compartment by electroosmosis through two ultrafiltration membranes. A mathematical model describing urea gradient as a function of operation parameters was established and its validity was examined by experiments. The longitudinal gradient of urea can be maintained through a suitable choice of operation parameters based on the simulation. Refolding of bovine serum by the urea gradient established in electric field and by direct oxidative dilution were performed, and evaluated by non-reductive SDS-PAGE and fluoresence spectrum, indicating the structural similarity between the BSA refolded in electric field to that of native BSA.