氨基己酸为配基的新型弱阳离子交换/疏水双功能色谱固定相对蛋白质的分离纯化

A novel dual-function weak cation exchange/hydrophobic interaction chromatography stationary phase with aminocaproic acid as the ligand for protein separation

以硅胶为基质、氨基己酸为配基制备了一种新型弱阳离子交换/疏水(WCX/HIC)双功能混合模式色谱固定相。该固定相配基具有一定的疏水性且含有羧基,在高盐浓度下表现为HIC的性质,可作为HIC固定相使用;在低盐浓度条件下表现为离子交换的性质,可作为WCX固定相使用。分别考察了该介质在WCX和HIC两种模式下对标准蛋白质的分离性能,并与商品柱进行比较。结果表明,所合成的WCX/HIC双功能固定相在WCX和HIC两种模式下对蛋白质均有较高的分离度和选择性,且分离能力与商品柱相当,两种模式下标准蛋白质的质量和活性回收率均大于93%,表明该柱具有"一柱二用"的功能,适于生物大分子的分离纯化。基于此双功能色谱柱构建的在线单柱二维液相色谱(2DLC-1C)可在60 min内实现8种蛋白质的快速分离。在70 min内完成了对蛋清中溶菌酶的二维纯化,纯度可达到98.3%。该技术中一根色谱柱可当作两根色谱柱使用,对蛋白质组学研究和重组蛋白药物的生产具有重要的应用价值。

A novel dual-function mixed-mode stationary phase based on silica gel functionalized with aminocaproic acid as the ligand was prepared. Because of the ligand with hydrophobicity and containing carboxyl function groups, it could display hydrophobic interaction chromatography (HIC) character in a high salt concentration mobile phase and weak cation exchange chromatography (WCX) character in a low salt concentration mobile phase. As a result, it could be employed to separate proteins with both WCX and HIC modes. The resolution and selectivity of the stationary phase were evaluated under both HIC and WCX modes with protein standards. In comparison with the conventional WCX and HIC columns, the results were satisfactory and acceptable. Protein mass and bioactivity recoveries of more than 93% could be achieved in both WCX and HIC mode using this column. The results indicated that the novel dual-function mixed-mode column in many cases could replace the use of two individual WCX and HIC columns. Based on this two-dimensional (2D) column, a new two-dimensional liquid chromatography (2DLC) technology with a single column (2DLC-1C) was developed. Eight kinds of protein standards could be separated completely with online 2DLC-1C within 60 min. It was also applied to purify lysozyme from egg white successfully with a purity of 98. 3%. It is important to proteome research and recombinant protein drug production for saving column expense and simplifying the process in biotechnology.