关键词: 置换色谱, 共聚焦激光扫描显微镜, 绿色荧光蛋白, 介质内传质, 肌醇六磷酸钠

Abstract: 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.

Key words: displacement chromatography, confocal laser scanning microscopy, green fluorescent protein, intraparticle mass transfer, phytic acid sodium salt hydrate