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化工学报  2013 

离子交换介质内蛋白置换行为的观测与解析

DOI: 10.3969/j.issn.0438-1157.2013.01.039, PP. 340-345

Keywords: 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

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Abstract:

以肌醇六磷酸钠(PAS)为置换剂研究了绿色荧光蛋白(eGFP)在QSepharoseHP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。

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