Affinity regulation of proteins with thermoresponsive polymer-brush interfaces

在本研究中，我们旨在制备一种新型的热响应界面来调节固定化金属离子对生物活性蛋白的亲和调节。为了实现这一目标，我们用亚氨基二乙酸（IDA）制备了热响应性聚合物接枝表面。然后，我们将这种热敏聚合物引入清洁的玻璃盖上，并研究了热敏溶菌酶的吸附。溶菌酶的氨基酸序列中只含有一个组氨酸（His）残基。因此，与镍离子共轭的ida通过配位键与His残基相互作用。由于聚合物链的水合作用，这种键很容易通过降低温度而断裂。事实上，溶菌酶在〓℃下通过ida -镍离子偶联物吸附在疏水的PIPAAm表面，而大多数吸附蛋白在不使用螯合剂或咪唑添加剂的情况下从PIPAAm表面自发解吸。这一结果为这种表面的制备提供了希望，这导致我们用硝基三乙酸（NTA）残基制备了PIPAAm表面。NTA目前用于纯化带有his标签的基因产物（即蛋白质）。然而，这种系统仍然需要螯合剂或咪唑作为标记蛋白的洗脱剂。然后，我们通过氨解PIPAAm衍生物在玻璃表面的叔丁基侧链引入NTA基团。这些表面在水环境中也表现出温度响应的溶菌酶吸附/解吸现象。综上所述，我们只能通过温度调节来调节His-tag蛋白的吸附/解吸行为。该系统在His标签蛋白分离中的应用还需要进一步的改进，在本项目中我们获得了很好的结果。我们将继续这项工作。

In this research, we aimed to prepare novel thermoresponsive interfaces modulating immobilized metal ion affinity regulation of bioactive proteins. To realize this aim, we prepared thermoresponsive polymer-grafted surfaces with iminodiacetic acid (IDA) . We then introduced such thermoresponsive polymers on cleaned glass coverslips and investigated thermoresponsive lysozyme adsorption. Lysozyme contains only one histigine (His) residue in its amino acid sequences. Thus, IDA-conjugated with nickel ions interacts with His residues through cordinated bonds. Such bonds are easily broken by reducing temperature because hydrated polymer chains. In fact, Lysozyme adsorbed on hydrophobized PIPAAm surfaces with IDA-nickel ion conjugate at 〓℃, while majority of adsorbed proteins spontaneously desorbed from the PIPAAm surfaces without using chelating agents or imidazole adition. This results is promissing for preparation of such surfaces which led us to prepare PIPAAm surfaces with nitrilotriacetic acid (NTA) residues. NTA is currently used in purification of gene products (i.e., proteins) with His-tag. However, such systems still needs chelating agents or imidazole for eluting agent of tagged proteins. Then, we introduced NTA groups through aminolysis of tert-butyl side chains of PIPAAm derivatives on glass surfaces. These surfaces also showed temperature responsive lysozyme adsorption/desorption phenomena in aqueous milieu. In conclusion, we can modulate His-tag protein adsorption/desorption behavior only through temperature modulation. We still needs to improve such systems for application in His tag protein separation, we obtained promissing results in this project. We will continue this work..