Interdisciplinary Biochemistry Graduate Program and Department of Chemistry, Indiana University, Bloomington, Indiana 47405, USA.
Biomacromolecules. 2009 Jul 13;10(7):1939-46. doi: 10.1021/bm900339q. Epub 2009 May 19.
A novel method to produce protein polymer conjugates for protein-acrylamide hydrogel formation is described. Alkenes are incorporated onto the N-terminus of expressed proteins to produce polymerizable protein monomers that can be utilized in protein-acrylamide copolymerization. A 4-vinylbenzoic acid thioester was synthesized and attached to the N-termini of two protein models, the immunoglobulin-binding protein Protein G and the bacterial enzyme xanthine-guanine phosphoribosyltransferase (GPRT), utilizing native chemical ligation. N-terminal cysteine containing proteins utilized in native chemical ligation reactions were generated from His-tagged fusion proteins using tobacco etch virus NIa (TEV) protease cleavage. The 4-vinylbenzyl functionalized proteins were good substrates for immobilizing proteins into polyacrylamide hydrogels via free radical induced protein-acrylamide copolymerization. The protein copolymerization procedures developed in this report are mild enough to allow proteins to retain measurable biological activity as demonstrated by the retention of immunoglobulin binding ability by immobilized Protein G and enzymatic activity of immobilized GPRT.
描述了一种用于蛋白质-丙烯酰胺水凝胶形成的蛋白质聚合物缀合物的新方法。在表达的蛋白质的 N 末端掺入烯烃以产生可用于蛋白质-丙烯酰胺共聚的聚合性蛋白质单体。合成了 4-乙烯基苯甲酸硫酯，并利用天然化学连接将其连接到两种蛋白质模型的 N 末端，即免疫球蛋白结合蛋白 Protein G 和细菌酶黄嘌呤-鸟嘌呤磷酸核糖基转移酶（GPRT）。在天然化学连接反应中使用的含 N 端半胱氨酸的蛋白质是使用烟草蚀纹病毒 NIa（TEV）蛋白酶切割 His 标记融合蛋白产生的。4-乙烯基苄基功能化蛋白质是通过自由基诱导的蛋白质-丙烯酰胺共聚将蛋白质固定在聚丙烯酰胺水凝胶中的良好底物。本报告中开发的蛋白质共聚程序足够温和，允许蛋白质保留可测量的生物活性，这可以通过固定化 Protein G 的免疫球蛋白结合能力和固定化 GPRT 的酶活性保留来证明。
