Design and Construction of Glutamine Binding Proteins with a Self-Adhering Capability to Unmodified Hydrophobic Surfaces as Reagentless Fluorescence Sensing Devices

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• 作者：Akira Wada ; Masayasu Mie ; Masuo Aizawa ; Pedro Lahoud ; Anthony E. G. Cass ; and Eiry Kobatake
• 刊名：Journal of the American Chemical Society
• 出版年：2003
• 出版时间：December 31, 2003
• 年：2003
• 卷：125
• 期：52
• 页码：16228 - 16234
• 全文大小：110K
• 年卷期：v.125,no.52(December 31, 2003)
• ISSN：1520-5126

文摘

The chemically and genetically remodeling of proteins with ligand binding specificities can beutilized to synthesize various protein-based microsensors for detecting single biomolecules. Here, wedescribe the construction and characterization of fluorophore-labeled glutamine binding proteins (QBP)and derivatives coupled to the independently designed hydrophobic polypeptide (E12) that can adhereonto solid surfaces via hydrophobic interactions. The single cysteine mutant (N160C QBP) modified withthe three environmentally sensitive fluorescent dyes (IAANS, acrylodan, and IANBD ester) showed increasedchanges in fluorescence intensity induced by glutamine binding. The use of these conjugates as reagentlessfluorescence sensors enables us to determine the glutamine concentrations (0.1-50 ges/entities/mgr.gif">M) in homogeneoussolution. The fusion of N160C QBP with E12, (Gly₄-Ser)n spacers (GSn), and IANBD resulted in the novelfluorescence sensing elements having an adhering capability to hydrophobic surfaces of unmodifiedmicroplates. In ELISA and fluorescence experiments for the microplates treated with a series of theconjugates, IANBD-labeled N160C QBP-GS1-E12 displayed the best reproducibility in adhesion onto thehydrophobic surfaces and the precise correlation between fluorescence changes and glutamine concentrations. The performance of the biosensor-attached microplate for glutamine titrations demonstrated that thehydrophobic interaction of E12 with solid surfaces is useful for effective immobilization of proteins thatneed specific conformational movements in recognizing particular biomolecules. Therefore, the techniqueusing E12 as a surface-linking domain for protein adhesion onto unmodified substrates could be appliedeffectively to prepare microplates/arrays for a wide variety of high-throughput assays on chemical andbiological samples.