Abstract

A new ion-exchange medium was prepared from magnetic particles of ∼1.0 µm through coating with small zwitterions and then functionalizing with ampholytic groups for the isoelectric point of ∼6.4 and denoted MSP-ZEWB. With Meyerozyma guilliermondii uricase (MGU) as the model of soluble proteins, the purification of a protein via ion-exchange was compared between MSP-ZEWB through elution in discontinuous mode and Toyopearl SP-650C as a classical ion-exchange medium through elution in continuous mode. MGU was adsorbed at pH 7.6 or 8.0 and eluted via competitive displacement by NaCl or electrostatic repulsions with an elution buffer at pH 10 to reverse the type of net charges of MGU. From MSP-ZEWB, MGU was eluted more rapidly with the elution percentages higher than those from Toyopearl SP-650C. For yielding a unit of MGU activity, MSP-ZEWB gave the elution solution volumes that were ∼50% of those obtained with Toyopearl SP-650C. The yields of MGU of the highest purity from MSP-ZEWB were higher than those from Toyopearl SP-650C, but the highest purification folds with both media were comparable. MSP-ZEWB regenerated for 16 times still showed the consistent purification efficacy. Therefore, the ion-exchange media bearing small zwitterion coats showed great promise for the purification of soluble proteins.

摘要

一种新的离子交换介质，它是由〜1.0 µm的磁性粒子通过涂有小的两性离子而制成的，然后用两性基团进行官能化，使等电点约为6.4，表示为MSP-ZEWB。以古细菌耶尔酵母（Myerozyma guilliermondii）尿酸酶（MGU）作为可溶性蛋白质的模型，通过比较了通过不连续模式洗脱的MSP-ZEWB和通过连续模式洗脱作为经典离子交换介质的Toyopearl SP-650C之间的离子交换。MGU吸附在pH 7.6或8.0上，并通过NaCl竞争性置换或静电排斥与pH 10的洗脱缓冲液洗脱，以反转MGU的净电荷类型。从MSP-ZEWB中洗脱MGU的速度比从Toyopearl SP-650C中洗脱的速度更快。为了产生一个单位的MGU活性，MSP-ZEWB的洗脱液体积约为用Toyopearl SP-650C获得的洗脱液体积的50％。MSP-ZEWB的最高纯度的MGU的产量高于Toyopearl SP-650C的最高，但两种培养基的最高纯化倍数相当。再生16次的MSP-ZEWB仍显示出一致的纯化功效。因此，带有小的两性离子涂层的离子交换介质显示了可溶蛋白纯化的巨大希望。