Kalia Jeet, Abbott Nicholas L, Raines Ronald T
Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706-1544, USA.
Bioconjug Chem. 2007 Jul-Aug;18(4):1064-9. doi: 10.1021/bc0603034. Epub 2007 May 9.
Protein microarrays are playing an increasingly important role in the discovery and characterization of protein-ligand interactions. The uniform orientation conferred by site-specific immobilization is a demonstrable advantage in using such microarrays. Here, we report on a general strategy for fabricating gold surfaces displaying a protein in a uniform orientation. An azido group was installed at the C-terminus of a model protein, bovine pancreatic ribonuclease, by using the method of expressed protein ligation and a synthetic bifunctional reagent. This azido protein was immobilized by Staudinger ligation to a phosphinothioester-displaying self-assembled monolayer on a gold surface. Immobilization proceeded rapidly and selectively via the azido group. The immobilized enzyme retained its catalytic activity and was able to bind to its natural ligand, the ribonuclease inhibitor protein. This strategy provides a general means to fabricate microarrays displaying proteins in a uniform orientation.
蛋白质微阵列在蛋白质-配体相互作用的发现和表征中发挥着越来越重要的作用。位点特异性固定所赋予的均匀取向是使用此类微阵列的一个明显优势。在此，我们报道了一种制备以均匀取向展示蛋白质的金表面的通用策略。通过表达蛋白连接法和一种合成双功能试剂，在模型蛋白牛胰核糖核酸酶的C末端安装了一个叠氮基。该叠氮基蛋白通过施陶丁格连接反应固定到金表面上展示膦硫酯的自组装单分子层上。固定反应通过叠氮基快速且选择性地进行。固定化的酶保留了其催化活性，并且能够结合其天然配体核糖核酸酶抑制蛋白。该策略提供了一种制备以均匀取向展示蛋白质的微阵列的通用方法。
