Site-directed Covalently Bound Fusion Protein Microarray

描述（由申请人提供）：生物芯片已成为基因表达，高通量筛选和药物发现的工具。虽然DNA芯片的使用迅速扩大，但蛋白质生物芯片尚未完善。原因之一是蛋白质固定的复杂性。蛋白质微阵列的潜在应用有很多，包括筛选蛋白质-蛋白质相互作用、检测翻译后修饰和筛选蛋白质-药物相互作用。一个系统，方便地固定蛋白质在一个受控的，定点的方式进行微阵列分析，而不需要荧光标记，将有助于填补一个重要的未满足的需求。

DESCRIPTION (provided by applicant): Biochips have become instrumental in gene expression, high throughput screening and drug discovery. While use of DNA chips has rapidly expanded, protein biochips have yet to be perfected. One reason is the complexity of protein immobilization. Potential applications of protein microarrays are numerous, including screening of protein-protein interactions, detection of post-translational modifications, and screening of protein-drug interactions. A system for conveniently immobilizing proteins in a controlled, site-directed manner for microarray analysis without the need for fluorescent labels would help fill a significant unmet need. The objective of this Phase I proposal is to establish the technical merit and feasibility of covalent, oriented attachment of proteins using a novel mutant enzyme capable of covalent binding to alkyl halides. This mutant enzyme and coupling chemistry were recently developed by a major supplier of biological, molecular and cell biology products. The proposed technology combines this enzyme technology with GenTel's highly controlled and well-characterized surface chemistry on gold and diamond surfaces, and surface plasmon resonance imaging (SPRi), an emerging microarray-compatible label-free detection method. Specific goals of this proposal include (1) optimization of linkers useful for covalent binding of the mutant enzyme to solid surfaces, (2) attachment of the linker and enzyme to the surface, (3) control of non-specific binding on the surface, and (4) detection of a model protein-protein interaction using SPRi. Using this approach, the direct immobilization of proteins from cell-free translation extracts for use in protein microarrays on gold (for SPRi detection) and novel diamond biochip surfaces (for fluorescent detection) can be accomplished. Phase II objectives will include in-vitro translation of these fusion proteins in cell-free extracts, direct immobilization from crude extracts, inclusion of biologically relevant protein-protein interactions, and development of SPR-compatible microfluidics.