Development of New concept for the evaluation of protein-or gene-function

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Objective of the research is to develop new concepts for the evaluation of protein-or gene-functions. In this term, following four categories were investigated ; 1 : protein immobilized biosensor, 2 : intracellular signal-detecting system using 2D-SPR biochip, 3 : intracellular signal-evaluating system using mass spectrometry, 4 : Theoretical consideration of SPR on micro-corn, In the protein-immobilized biosensor, thyroid hormon receptor (TR) and dioxin receptor (AhR) were immobilized on a gold electrode through oligohistidine-tag using nickel-NTA complex. These sensors responded to their ligands including thyroxine, naphthoflavon and benzopyrene. The detection sensitivities of these sensors were comparable to those of Rl methods, In the 2D-SPR system, specific substrate of protein kinase A, protein kinase C and Src were immobilized on the gold surface at the thiol group in the N-terminus-cysteine residue using maleimide group on the surface. obtained chip was imaged with 2D-SPR imager using anti-phosphotyrosin antibody. Phosphorylation reaction with protein kinase could be successfully monitored using this system. This system can be a good method for the profiling of intracellular signals. In the mass spectrometric system, the change of PKA activity could be evaluated using PC-12 cell after the treatment of various drugs. This results were compared with those of reporter gene assay. Both results had good feasibility. This means that his method can be used as the evaluation system of cellular function. PKC and Src Activities were also able to be monitored using this system. In the investigation of the SPR sensor using micro-corn, theoretical consideration was investigated and the theory of SPR phenomena on such microarea could be established.