Affinity regulation of proteins with thermoresponsive polymer-brush interfaces

在这项研究中，我们的目标是制备新型的热响应界面来调节生物活性蛋白的固定化金属离子亲和力。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，我们将这种热敏性聚合物引入清洁的玻璃盖片上，并研究了热敏性溶菌酶的吸附。溶菌酶的氨基酸序列只有一个组氨酸(His)残基。因此，IDA-与镍离子的结合通过配位键与His残基相互作用。这样的键很容易通过降低温度而断裂，因为水合的聚合物链。事实上，在〓℃时，溶菌酶以IDA-镍离子结合物的形式吸附在疏水的PIPAAm表面，而大多数吸附的蛋白质在没有使用螯合剂或咪唑的情况下自发地从PIPAAm表面解吸出来。这一结果为制备这类表面提供了有利条件，这使得我们用氮三乙酸(NTA)残基制备了PIPAAM表面。NTA目前用于纯化带有组氨酸标签的基因产物(即蛋白质)。然而，这类系统仍然需要使用螯合剂或咪唑作为标记蛋白的洗脱剂。然后，我们通过PIPAAm衍生物的叔丁基侧链在玻璃表面的氨解引入了NTA基团。在水环境中，这些表面还表现出对温度敏感的溶菌酶吸附/解吸现象。综上所述，我们只能通过温度调节来调控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进一步完善，本项目取得了可喜的成果。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..