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.
Thesaurus Terms: high throughput technology, hydrolase, immobilized enzyme, microarray technology, mutant, protein binding, protein quantitation /detection, technology /technique development alkyl group, covalent bond, gold, protein protein interaction, surface property biotechnology, cell free system, recombinant protein, site directed mutagenesis, surface plasmon resonance
