Department of Chemical and Biomolecular Engineering, Clemson University, 127 Earle Hall, Clemson, SC 29634, USA.
J Chromatogr A. 2010 Jul 23;1217(30):4946-57. doi: 10.1016/j.chroma.2010.05.049. Epub 2010 Jun 2.
This contribution describes the purification of anthrax protective antigen (PA) protein from Escherichia coli lysate using bind-and-elute chromatography with newly designed weak anion-exchange membranes. Protein separation performance of the new AEX membrane adsorber was compared with the commercial Sartobind D membrane adsorber and HiTrap DEAE FF resin column under preparative scale conditions. Dynamic protein binding capacities of all three stationary phases were determined using breakthrough curve analysis. The AEX membrane showed higher binding capacities than the Sartobind D membrane at equivalent volumetric throughput and higher capacities than the HiTrap DEAE FF resin column at 15 times higher volumetric throughput. Anion-exchange chromatography was performed using all three stationary phases to purify PA protein. Quantitative SDS-PAGE analysis of effluent fractions showed that the purity of PA protein was higher for membrane adsorbers than the HiTrap DEAE FF resin column and was the same for the new AEX membrane and Sartobind D membrane adsorbers. The effects of E. coli lysate load volume and volumetric flow rate on PA protein separation resolution using the membrane adsorbers were minor, and the peak elution profile remained un-changed even under conditions where >75% of the total protein dynamic binding capacity of the membranes had been utilized. PA protein peak resolution was higher using pH-gradient elution than with ionic strength gradient elution. Overall, the results clearly demonstrate that membrane chromatography is a high-capacity, high-throughput, high-resolution separation technique, and that resolution in membrane chromatography can be higher than resin column chromatography under preparative conditions and at much higher volumetric throughput.
本文描述了使用新型弱阴离子交换膜的绑定-洗脱色谱法从大肠杆菌裂解物中纯化炭疽保护性抗原（PA）蛋白。在制备规模条件下，比较了新的 AEX 膜吸附剂与商业 Sartobind D 膜吸附剂和 HiTrap DEAE FF 树脂柱的蛋白质分离性能。使用穿透曲线分析确定了所有三种固定相的动态蛋白质结合容量。与 Sartobind D 膜相比，AEX 膜在等效体积通量下具有更高的结合容量，与 HiTrap DEAE FF 树脂柱相比，在 15 倍更高的体积通量下具有更高的容量。使用所有三种固定相进行阴离子交换色谱法来纯化 PA 蛋白。流出部分的定量 SDS-PAGE 分析表明，膜吸附剂的 PA 蛋白纯度高于 HiTrap DEAE FF 树脂柱，与新型 AEX 膜和 Sartobind D 膜吸附剂相同。膜吸附剂对 PA 蛋白分离分辨率的大肠杆菌裂解物加载量和体积流速的影响较小，即使在膜的总蛋白质动态结合容量的>75%已被利用的情况下，峰洗脱曲线仍保持不变。与离子强度梯度洗脱相比，pH 梯度洗脱的 PA 蛋白峰分辨率更高。总的来说，结果清楚地表明，膜色谱法是一种高容量、高通量、高分辨率的分离技术，并且在制备条件下和更高的体积通量下，膜色谱法的分辨率可以高于树脂柱色谱法。
