Site-specific protein immobilization through N-terminal oxime linkages
Site-specific protein immobilization through N-terminal oxime linkages
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DOI：
10.1039/b618002g
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发表时间：
2007-01-01
影响因子：
--
通讯作者：
Maynard, Heather D.
中科院分区：
文献类型：
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作者：
Christman, Karen L.;Broyer, Rebecca M.;Maynard, Heather D.
将蛋白质固定在特定方向对于诊断蛋白质阵列、生物材料和其他需要保留生物活性的应用非常重要。我们报告了一种蛋白质微图案化方法，该方法利用化学选择性反应将 N 末端的蛋白质缀合到聚合物薄膜上。通过自由基聚合合成了甲基丙烯酸2-羟乙酯和Boc保护的氨氧基四(乙二醇)甲基丙烯酸酯的共聚物。使用基于光致产酸剂的光刻法对 Boc 基团进行局部脱保护。使用绿色荧光醛微球通过荧光显微镜验证微图案。经过氨基转移反应以在 N 末端安装 α-酮酰胺基团的链霉亲和素通过形成肟键与图案缀合。由于大多数蛋白质可能被修饰以含有反应性羰基，因此该方法应该适用于特别是通过 N 末端对多种蛋白质进行图案化。
Immobilizing proteins in specific orientations is important for diagnostic protein arrays, biomaterials, and other applications where retention of bioactivity is essential. We report an approach for protein micropatterning that exploits a chemoselective reaction to conjugate proteins at the N-terminus to polymer films. A copolymer from 2-hydroxyethyl methacrylate and a Boc-protected aminooxy tetra( ethylene glycol) methacrylate was synthesized by radical polymerization. Boc groups were locally deprotected using photoacid generator-based photolithography. Micropatterns were verified by fluorescence microscopy utilizing green fluorescent aldehyde microspheres. Streptavidin that was subjected to a transamination reaction to install an alpha-ketoamide group at the N-terminus was conjugated to the patterns by oxime bond formation. Since the majority of proteins may be modified to contain a reactive carbonyl group, this methodology should be applicable to pattern a wide variety of proteins specifically through the N-terminus.