Innovative Modular Membrane Adsorber System for High‐Throughput Downstream Screening for Protein Purification
Innovative Modular Membrane Adsorber System for High‐Throughput Downstream Screening for Protein Purification
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创新的模块化膜吸附器系统，用于蛋白质纯化的高通量下游筛选
DOI：
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发表时间：
2006
期刊：
影响因子：
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通讯作者：
R. Ulber
中科院分区：
文献类型：
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作者：
Öznur Kökpinar;D. Harkensee;C. Kasper;T. Scheper;R. Zeidler;O. Reif;R. Ulber
为了开发最有效的蛋白质纯化策略，设计并测试了两种具有不同功能的吸附器膜装置：8条和单旋柱。在微升尺度上可以同时确定从不同生物基质中分离出多个目标蛋白的最适宜的膜吸附器类型和最佳的层析吸附/洗脱条件。对离子交换(IEX)、金属络合物(MC)和刀豆蛋白A(ConA)修饰的膜类型进行了测试。以牛血清白蛋白(BSA)和溶菌酶为模型蛋白，考察了8-带阴离子交换膜和阳离子交换膜的结合能力和蛋白质回收率。分别从发酵液和裂解液中分离His6标记的蛋白质BGL-His和GFP-His。研究了三元蛋白质混合物(BSA、溶菌酶和BGL-His)在8条IEX和金属螯合膜层析中的分离行为。在负载Cu2+的金属螯合膜自旋柱的基础上，以葡萄糖氧化酶(GOD)为模型蛋白，研制了ConA亲和装置。综上所述，膜吸附技术具有处理速度快、易于放大等优点。这些设备可以直接在膜上装入预先澄清的发酵液或细胞裂解液，并通常只需一步就能分离出感兴趣的蛋白质。
To develop the most efficient strategy for the purification of proteins, two types of adsorber membrane devices with different functionalities were designed and tested: 8‐strips and single spin columns. The most suitable type of membrane adsorber and the optimal chromatographic loading/elution conditions for several target proteins from different biological matrices could be determined simultaneously in microliter scale. Ion exchange (IEX), metal chelate (MC), and Concanavalin A (Con A) modified membrane types were tested in the devices. Bovine serum albumin (BSA) and lysozyme were used as model proteins for investigations of the binding capacity and protein recovery percentage of the 8‐strip anion exchange and the cation exchange membrane. The isolation of His6‐tagged proteins, Bgl‐His and GFP‐His from fermentation broth and lysate, respectively, was performed using an 8‐strip metal chelate affinity membrane loaded with different metal ions. Separation behavior of a ternary protein mixture (BSA, lysozyme, and Bgl‐His) was studied in 8‐strips IEX and metal chelate membrane chromatography. The Con A affinity devices were developed on the basis of metal chelate membrane spin columns loaded with Cu2+ ions and investigated using glucose oxidase (GOD) as model protein. In summary, the advantages of the membrane adsorber technology, such as fast processing and easy scale‐up, were utilized. The devices made it possible to load the membrane directly with preclarified fermentation broth or cell lysate and separate the protein of interest often in a single step.