Selective immobilization of proteins to self-assembled monolayers presenting active site-directed capture ligands
Selective immobilization of proteins to self-assembled monolayers presenting active site-directed capture ligands
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DOI：
10.1073/pnas.072685299
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发表时间：
2002-04-16
影响因子：
11.1
通讯作者：
Mrksich, M
中科院分区：
文献类型：
--
作者：
Hodneland, CD;Lee, YS;Mrksich, M
本文描述了一种通过控制蛋白质的密度和取向，将蛋白质选择性和共价固定在表面的方法。该策略基于丝氨酸酯酶角质化酶与呈现膦酸配体的自组装单层的结合，以及随后将配体与酶活性位点共价结合的置换反应。表面等离子体共振（SPR）光谱分析表明，表皮酶不可逆地与一层单层结合，在三（乙二醇）基团中以1%的密度呈现捕获配体。共价固定化对角质酶是特异性的，乙二醇端接的单层有效地防止了蛋白质的非特异性吸附。为了证明该方法可以用于固定感兴趣的蛋白，构建了一个角质酶-钙调蛋白融合蛋白并将其固定在单层上。SPIR显示钙调蛋白与固定化钙调蛋白选择性结合。这种捕获配体固定方法结合了固定反应对目标蛋白具有高选择性，系链是共价的，因此稳定的优点，并且该方法避免了在固定前对蛋白质进行合成修饰和严格纯化的需要。这些特点使该方法非常适合于一系列应用，特别是用于构建蛋白质微阵列。
This paper describes a method for the selective and covalent immobilization of proteins to surfaces with control over the density and orientation of the protein. The strategy is based on binding of the serine esterase cutinase to a self-assembled monolayer presenting a phosphonate ligand and the subsequent displacement reaction that covalently binds the ligand to the enzyme active site. Surface plasmon resonance (SPR) spectroscopy showed that cutinase binds irreversibly to a monolayer presenting the capture ligand at a density of 1 % mixed among tri(ethylene glycol) groups. The covalent immobilization is specific for cutinase, and the glycol-terminated monolayer effectively prevents unwanted nonspecific adsorption of proteins. To demonstrate that the method could be used to immobilize proteins of interest, a cutinase-calmodulin fusion protein was constructed and immobilized to the monolayer. SPIR showed that calcineurin selectively associated with the immobilized calmodulin. This capture ligand immobilization method combines the advantages that the immobilization reaction is highly selective for the intended protein, the tether is covalent and, hence, stable, and the method avoids the need for synthetic modification and rigorous purification of proteins before immobilization. These characteristics make the method well suited to a range of applications and, in particular, for constructing protein microarrays.