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

谷氨酸脱羧酶的大规模生产、纯化及其在糖尿病诊断中的应用

• 批准号: 10556018
• 负责人: UENO Hiroshi
• 金额: $ 5.31万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10556018/
• 关键词: decarboxylase; GABA; diabetes; diagnosis

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.

利用酿酒酵母构建了大鼠脑谷氨酸脱羧酶同工酶GAD65的表达系统。GAD65被发现是I型糖尿病患者产生自身抗体的靶蛋白。这种自身抗体存在于糖尿病发展的早期阶段，早在糖尿病症状出现前10年就存在。因此，我们的目标是获得大量可用于糖尿病诊断的GAD65。我们通过改变碳源、替换启动子和终止子序列、改变诱导材料等方法优化了生长条件。我们检测了表达的蛋白质的热稳定性，其中我们发现竞争性抑制剂显著保护蛋白质免受热灭活。提供稳定的酶是开发诊断试剂的要求之一。我们还研制了一种简便、微量的酶活性测定仪。微毛细管和光控计数器的结合给出了令人满意的结果。我们目前正在努力优化设备。

项目成果

Ueno H.: "Enzymatic and structural aspects on glutamate decarboxylase"J. Mol. Catalysis B: Enzymatic. (in press). (2000)

Ueno H.：“谷氨酸脱羧酶的酶学和结构方面”J。

Ueno,H.: "Enzymatic and structural aspects on gluamate decarboxylase"J. Mol. Catalysis B : Enzymatic. (in press). (2000)

Ueno，H.：“谷氨酸脱羧酶的酶学和结构方面”J。

Ueno, H.: "Enzymatic and structural aspects on glutamate decarboxylase"J. Mol. Catalysis B : Enzymatic. (in press). (2000)

Ueno, H.：“谷氨酸脱羧酶的酶学和结构方面”J。

Ueno,H.: "Expression of human histidine decarboxy lase in Saccharomyces cerevisiae"J. Biochem. Mol. Biol. Biophys.. 2(2). 141-146 (1998)

Ueno，H.：“人组氨酸脱羧酶在酿酒酵母中的表达”J。

Ueno, H.: "Expression of human histidine decarboxylase in Saccharomyces cerevisiae"J. Biochem. Mol. Biol. Biophys.. 2. 141-146 (1998)

Ueno，H.：“人组氨酸脱羧酶在酿酒酵母中的表达”J。