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Secretion of human proteins by bacteria and conformational change of protein during secretion

细菌分泌人类蛋白质及分泌过程中蛋白质的构象变化

基本信息

批准号：
02403024
负责人：
UDAKA Shigezo
金额：
$ 12.16万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (A)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1992
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02403024/
关键词：
Protein production Secretion of human proteins Conformational change during secretion

项目摘要

By using a protein-production system with Bacillus brevis as a host, human proteins can be efficiently produced, although its efficiency is much lower than that of bacterial protein-production with some exception. Since it is presumed that the production efficiency depends on the structural change of the protein during its synthesis and secretion, we studied the enzymes con- trolling the formation of tertiary structure of proteins, i.e., peptidylprolyl cis-trans isomerase (PPI), protein disulfide isomerase (PDI), and proteases which discriminate the protein degradation according to its structure.We found PPI activities from culture supernatant and cell extract of B. brevis, from which the enzyme was partially purified and its properties were analyzed. This enzyme is relatively heat stable and its activity was lowered by increasing salt concentrations of the reaction mixture. It was not sensitive to either cyclosporin A or FK506, an immunosuppressant. PDI like enzyme, i.e., protein disulfide bond forming enzyme (DSB), was found only in the culture supernant of B. brevis. We succeeded to clone the DSB gene by a shot-gun method. The nucleotide sequence of the gene revealed that the molecular weight of DSB is about 13,000 and it has the sequence of signal peptide and CysGlyTyr- Cys, which is the catalytic site of the PDI like enzyme. These secreted enzymes are presumed to play important roles in the formation of foreign proteins having correct conformation and fully biological activity.

通过使用以短芽孢杆菌为宿主的蛋白质生产系统，可以有效地生产人类蛋白质，尽管其效率比细菌蛋白质生产的效率低得多，但有一些例外。由于假定生产效率取决于蛋白质在其合成和分泌过程中的结构变化，我们研究了控制蛋白质三级结构形成的酶，即，肽基脯氨酰顺反异构酶（PPI）、蛋白质二硫键异构酶（PDI）和根据蛋白质结构区分蛋白质降解的蛋白酶。对该酶进行了部分纯化，并对其性质进行了分析。该酶是相对热稳定的，并且其活性通过增加反应混合物的盐浓度而降低。对免疫抑制剂FK 506和环孢素A均不敏感。PDI样酶，即，蛋白质二硫键形成酶（DSB）仅存在于B的培养上清中。brevis。我们用鸟枪法成功地克隆了DSB基因。该基因的核苷酸序列显示，DSB的分子量约为13，000，它具有信号肽和CysGlyTyr-Cys的序列，这是PDI样酶的催化位点。这些分泌的酶被认为在形成具有正确构象和完全生物活性的外源蛋白质中起重要作用。