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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..

本研究旨在制备新型的温度响应性界面，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）制备了温敏聚合物接枝表面。然后，我们介绍了这种温敏聚合物清洁玻璃盖玻片和研究温敏溶菌酶吸附。溶菌酶在其氨基酸序列中仅含有一个组氨酸（His）残基。因此，与镍离子缀合的IDA通过协调键与His残基相互作用。由于聚合物链水合，这种键很容易通过降低温度而断裂。事实上，溶菌酶在20 ℃下吸附在IDA-镍离子结合的疏水化PIPAAm表面上，而大多数吸附的蛋白质在不使用螯合剂或咪唑添加剂的情况下自发地从PIPAAm表面脱附。这一结果是有希望的制备这样的表面，这导致我们准备PIPAAm表面与氨三乙酸（NTA）残基。NTA目前用于纯化基因产物（即，蛋白质）。然而，这样的系统仍然需要螯合剂或咪唑作为标记蛋白质的洗脱剂。然后，我们通过在玻璃表面上氨解PIPAAm衍生物的叔丁基侧链引入NTA基团。这些表面还显示出温度响应性的溶菌酶吸附/解吸现象在水环境中。总之，我们只能通过温度调节来调节His标签蛋白的吸附/解吸行为。本课题在His标签蛋白分离方面取得了令人满意的结果，但仍需进一步完善。我们将继续这项工作。