离子交换介质内蛋白置换行为的观测与解析

Visualizing and analyzing protein displacement in ion exchange adsorbent

以肌醇六磷酸钠(PAS)为置换剂研究了绿色荧光蛋白(eGFP)在Q Sepharose HP离子交换色谱(IEC)介质上的置换行为。置换色谱结果表明,不同浓度PAS下eGFP均可形成稳定的置换列并成功分离。利用共聚焦激光扫描显微镜(CLSM)技术对eGFP在离子交换色谱介质内吸附动力学的观测发现,eGFP在IEC介质内的吸附是由表面扩散和孔扩散共同作用的复杂行为。置换过程中不同时刻的CLSM图像显示,eGFP置换不仅取决于置换剂的亲和力,也与置换剂与介质的结合过程相关;介质内荧光强度的分析表明,eGFP置换首先发生在介质的外层,PAS浓度的降低导致置换速率下降。这与置换色谱的结果相吻合。CLSM不仅为蛋白置换动力学的研究提供了一种直观观测技术,CLSM结果也确认了蛋白质置换过程的复杂性。

The displacement behavior of enhanced green fluorescent protein（eGFP）on Q Sepharose HP was investigated using phytic acid sodium salt hydrate（PAS）as the displacer.The protein displacement in chromatographic column indicated that a stable displacement train of eGFP could be developed at different PAS concentrations,and subsequently a successful separation of eGFP was achieved.Furthermore,eGFP adsorption and displacement on the particle scale were also visualized by confocal laser scanning microscopy（CLSM）and further analyzed to elucidate their mechanism.CLSM images in protein adsorption exhibited that mass transfer of eGFP inside Q Sepharose HP was a complicated phenomenon controlled by both surface and pore diffusion.Moreover,CLSM images in the displacement process on the particle scale indicated that the displacement of eGFP by PAS not only depended on the affinity of the displacer but also was related to the kinetics of PAS binding.Finally,the trajectory of radial fluorescent distribution inside the adsorbent illustrated that eGFP displacement occurred firstly at the outmost layer of the adsorbent and a decrease in displacer concentration led to a drop of fluorescent fading rate.It was consistent with the results in displacement chromatography of eGFP.It was confirmed that CLSM provided an intuitive tool to visualize the intraparticle displacement of protein,and gained insight into the complexity of protein displacement at the particle scale.