琼脂糖－DEAE离子交换介质的配基密度和孔径对BSA吸附的影响

Influences of ligand density and pore size on BSA adsorption on agarose-based DEAE-ion-exchange resins

离子交换色谱是蛋白质分离纯化的有效方法之一,配基密度和介质孔径是影响蛋白质吸附的关键因素。采用3种不同琼脂糖浓度的凝胶为基质,具有不同的平均孔径,分别偶联上阴离子交换配基DEAE,通过调控反应条件,包括反应温度、反应时间、碱浓度和DEAE浓度,得到了不同配基密度和介质孔径的系列DEAE离子交换介质。考察了牛血清白蛋白(BSA)的静态和动态吸附性能,发现随配基密度增加或介质孔径减小,BSA饱和吸附容量有所增大;对于吸附动力学,介质孔径显著影响有效扩散系数。结果表明,配基密度和介质孔径共同决定了蛋白质的吸附性能,介质孔径主导蛋白质的孔内扩散,而配基密度则影响配基-蛋白质间的相互作用。

Ion exchange chromatography （IEC） is a common and powerful technique for the purification of proteins. The ligand density and pore size of the resins have significant effects on the separation behaviors of protein. In the present work, three kinds of crossed-linked agarose gel with different agarose concentrations were used as the matrices to represent various pore sizes, and anionic ligand, diethylaminothyl （DEAE）, was coupled with different ligand densities. The preparation conditions, including the reaction temperature, time, DEAE concentration and NaOH concentration, were optimized to control the ionic exchange capacity. A series of DEAE resins with different ligand densities and pore sizes were obtained. The adsorption isotherms and kinetics of bovine serum albumin （BSA） were measured on these resins. The saturated adsorption capacity increased with the increasing of ligand densities and the decreasing of pore sizes at the range tested. For the adsorption kinetics, the pore size influenced significantly the effective pore diffusivities. The results demonstrated that both the pore size and ligand density affect the static and dynamic adsorption. The pore size dominates the diffusion and mass transfer of protein, while the ligand density affects the interactions between ligand and protein.