Department of Chemistry, University of Richmond, Gottwald Center for the Sciences, 28 Westhampton Way, Richmond, Virginia 23173, USA.
Bioconjug Chem. 2009 Dec;20(12):2381-4. doi: 10.1021/bc900341s.
A robust method to immobilize a maltose biosensor is described using an engineered maltose periplasmic binding protein (PBP) covalently coupled to NBDamide, an environmentally sensitive fluorophore. A mesoporous silica sol-gel derived from diglycerylsilane (DGS) was constructed to embed the maltose biosensor, and the ligand reporting fluorescence properties were measured. When sequestered in the DGS-derived silica matrix, the biosensor retained maltose-dependent fluorescence sensing capability with micromolar affinity, which is consistent with the protein free in solution. The MBP-NBD conjugate was further modified by covalent conjugation with poly(ethylene glycol)-5000 (PEG) to promote the retention of water molecules around the protein and to reduce possible steric effects between the silica matrix and protein. Bioconjugation with PEG molecules does not significantly affect the signaling response of the protein in solution. When immobilized in the DGS polymer, a consistent increase in fluorescence intensity was observed as compared to the protein not functionalized with PEG. To our knowledge, this report presents the first successful method to embed a PBP biosensor in a polymerized matrix and retain signaling response using an environmentally sensitive probe. The immobilization method presented here should be easily adaptable to all conformation-dependent biosensors.
描述了一种使用工程化的麦芽糖周质结合蛋白（PBP）与 NBDamide 共价偶联的方法，将麦芽糖生物传感器固定化，NBDamide 是一种对环境敏感的荧光团。采用二甘醇基硅烷（DGS）构建介孔硅溶胶-凝胶，用于嵌入麦芽糖生物传感器，并测量配体报告的荧光性质。当被隔离在 DGS 衍生的硅基质中时，生物传感器保留了麦芽糖依赖性的荧光传感能力，具有微摩尔亲和力，与游离在溶液中的蛋白质一致。MBP-NBD 缀合物通过与聚乙二醇-5000（PEG）的共价缀合进一步修饰，以促进蛋白质周围水分子的保留，并减少硅基质和蛋白质之间可能的空间位阻。PEG 分子的生物缀合不会显著影响蛋白质在溶液中的信号响应。与未用 PEG 功能化的蛋白质相比，在 DGS 聚合物中固定化时，观察到荧光强度的一致增加。据我们所知，本报告首次成功地将 PBP 生物传感器嵌入聚合基质中，并使用对环境敏感的探针保留信号响应。这里提出的固定化方法应该很容易适应所有构象依赖性生物传感器。
