Large production and purification of glutamate decarboxylase and application for diagnosing diabetes mellitus

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Expression system of an isozyme of rat brain glutamate decarboxylase, GAD65, was constructed with Saccharomyces cerevisiae. GAD65 is found to be a target protein for autoantibody produced in type I diabetic patients. This autoantibody exists in the early stages of diabetic development, as early as 10 year prior to showing the diabetic symptom. Therefore, our aim was to obtain a large amount of GAD65 which might be used in the diagnosis of the diabetes.We have optimized the growth conditions by varying the carbon source, replacing the promoter and terminator sequences, and altering the inducing materials. We have examined thermal stability of the expressed protein, in which we found that competitive inhibitors significantly protect the protein from the thermal inactivation. Providing a stable enzyme is one of the requirements for the development of diagnostic agents. We have also developed an apparatus for convenient assaying the enzymatic activity in minute quantity. Combination of micro-capillary tube and photo-operated counter gave promising results. We are currently working to optimize the apparatus.