Le Borgne S, Graber M, Condoret J S
Département de Génie Biochimique et Alimentaire, UA CNRS 544 INSA, Toulouse, France.
Bioseparation. 1995 Feb;5(1):53-64.
Poly(glutamic acid) tail consisting of 6 glutamate residues was fused to the N-terminus of Escherichia coli beta-galactosidase (beta-gal), by genetic engineering techniques. The wild-type and modified genes were expressed intracellularly and in soluble state in Escherichia coli, leading to the proteins respectively designated beta-gal2 and E6-beta-gal. Both enzymes were purified by affinity chromatography. The specific activity of purified E6-beta-gal was found to be comparable to the wild-type enzyme and its increased net charge was indicated by lon-Exchange Chromatography (IEC). The use of such a charged fusion for selective recovery of beta-gal from cell extract using IEC and Ion-Exchange Membrane Chromatography (IEMC) was explored. The additional charges enabled the separation factor to be increased about two-fold on both IEC and IEMC, but the IEMC step achieved a better throughput than the IEC step. The selectivity of recovery promoted by the charged tail was further analysed by processing the experimental data obtained in IEC with the Stoichiometric Displacement Model, a recent model very appropriate for the understanding of the retention of polymeric biomolecules on ion-exchangers. It was shown that E6-beta-gal had the same characteristic charge as beta-gal2 but that the binding constant to the ion-exchanger of the tagged beta-gal was 6 times greater than for the wild-type enzyme.
通过基因工程技术，将由6个谷氨酸残基组成的聚谷氨酸尾巴融合到大肠杆菌β-半乳糖苷酶（β-gal）的N端。野生型和修饰基因在大肠杆菌中进行细胞内表达且呈可溶状态，分别产生了命名为β-gal2和E6-β-gal的蛋白质。两种酶均通过亲和层析进行纯化。发现纯化后的E6-β-gal的比活性与野生型酶相当，离子交换色谱（IEC）显示其净电荷增加。探索了使用这种带电荷的融合蛋白通过IEC和离子交换膜色谱（IEMC）从细胞提取物中选择性回收β-gal的方法。额外的电荷使IEC和IEMC上的分离因子提高了约两倍，但IEMC步骤的通量比IEC步骤更高。通过用化学计量置换模型处理在IEC中获得的实验数据，进一步分析了带电荷尾巴促进回收的选择性，该模型是一种非常适合理解聚合生物分子在离子交换剂上保留情况的最新模型。结果表明，E6-β-gal与β-gal2具有相同的特征电荷，但标记的β-gal与离子交换剂的结合常数比野生型酶大6倍。
