Affinity regulation of proteins with thermoresponsive polymer-brush interfaces

具有热响应聚合物刷界面的蛋白质亲和力调节

基本信息

批准号：
18500367
负责人：
KIKUCHI Akihiko
金额：
$ 2.63万
依托单位：
Tokyo University of Science
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

项目摘要

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..

在这项研究中，我们旨在准备新型的热响应界面，以调节生物活性蛋白的固定金属离子亲和力调节。为了实现这一目标，我们用米诺二乙酸（IDA）准备了热响应性聚合物接枝表面。然后，我们在清洁的玻璃盖玻片上引入了这种热响应聚合物，并研究了热响应性溶菌酶的吸附。溶菌酶在其氨基酸序列中仅包含一个组织（HIS）。那是与镍离子相结合的，通过有绳粘结与他的残留物相互作用。由于水合聚合物链，这种键很容易通过降低温度而破裂。实际上，溶菌酶以iDa-nickel离子共轭物吸附在疏水的pipaam表面上，而大多数吸附的吸附蛋白在不使用螯合剂或咪唑版本的情况下从PIPAAM表面上吸收了蛋白质。目前，该结果用于用His-TAG纯化基因产物（即蛋白质）。但是，这种系统仍然需要螯合剂或咪唑才能洗脱标记蛋白的剂。然后，我们通过对玻璃表面上的Pipaam衍生物的TERT叔丁基侧链进行氨基溶解来引入NTA组。这些表面还显示了水环中温度响应溶菌酶的吸附/解吸现象。总之，我们只能通过温度调节来调节HIS-标签蛋白吸附/解吸行为。我们仍然需要改善此类系统以在其TAG蛋白分离中应用，我们在该项目中获得了希望的结果。我们将继续这项工作。