Studies on the folding process of the polypeptide after its translocation across the membrane

多肽跨膜易位后折叠过程的研究

• 批准号: 06660423
• 负责人: UDAKA Shigezo
• 金额: $ 1.34万
• 依托单位: Tokyo University of Agriculture
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06660423/
• 关键词: Bacillus brevis; Disulfide bond of protein; Protein folding; Protein secretion

We have shown that human proteins having several disulfide bonds in their molecules can be efficiently secreted by the Bacillus brevis host-vector system which we developed. In this system, a disulfide bond forming enzyme must have an important role in the formation of the correctly folded and biologically active protein after the translocation across the membrane of the unfolded polypeptide. So we cloned the gene (bdb) for protein thiol-disulfide exidoreductase (a disulfide bond forming enzyme) from Bacillus brevis that encodes a protein consisting of 117 amino acids with a signal peptide of 27 residues. Bdb contains a well-conserved motif, Cys-X-X-Cys, which functions as the active center of proteins such as protein disulfide isomerase and thioredoxin. The bdb gene complemented the Escherichia coli dsbA mutation, restoring motility by means of flagellar and alkaline phosphatase activity. The Bdb protein overproduced in B.brevis was enzymatically active in both reduction and oxidation of disulfide bonds in vitro. Immunoblotting indicated that Bdb could function at the periphery of the cell.

我们已经证明，我们开发的短杆菌宿主载体系统可以有效地分泌分子中含有几个二硫键的人类蛋白质。在这个系统中，二硫键形成酶必须在未折叠的多肽跨膜转运后形成正确折叠的具有生物活性的蛋白中发挥重要作用。因此，我们克隆了短芽孢杆菌硫醇二硫键形成酶(一种二硫键形成酶)的基因(Bdb)，它编码一个由117个氨基酸组成的蛋白质，一个27个残基的信号肽。BDB含有一个保守的基序Cys-X-X-Cys，它是蛋白质二硫键异构酶和硫氧还蛋白的活性中心。Bdb基因补充了大肠杆菌dsbA突变，通过鞭毛和碱性磷酸酶活性恢复了运动能力。在短杆菌中高表达的BDB蛋白在体外对二硫键的还原和氧化都具有酶活性。免疫印迹显示，BDB可以在细胞的外围发挥作用。

项目成果

T.Ishihara: "Cloning and characterization of the gene for a protein thiol-disulfide oxidoreductase in B.brevis" Journal of Bacteriology. 177. 745-749 (1995)

T.Ishihara：“短双歧杆菌中蛋白质硫醇二硫化物氧化还原酶基因的克隆和表征”细菌学杂志。

Tatsuya Ishihara et al.: "Cloning and characterization of the gene for a protein thilo-disulfide oxidoreducatase in Bacillus brevis" Journal of Bacteriology. Vol. 177. 745-749 (1995)

Tatsuya Ishihara 等人：“短芽孢杆菌中蛋白质硫代二硫键氧化还原酶基因的克隆和表征”细菌学杂志。

Tatsuya Ishihara et al.: "Cloning and Characterization of the gene for a protein thid-disultide oxidoveductase in Bacillus brevis" Journal of Bacteriology. 177. 745-749 (1995)

Tatsuya Ishihara 等人：“短芽孢杆菌中蛋白质二硫化物氧化酶基因的克隆和表征”细菌学杂志。

Shigezo Udaka: "Protein secretion in Bacillus brevis." Antonie van Leeuwenhoek. Vol. 64. 137-143 (1993)

Shigezo Udaka：“短芽孢杆菌的蛋白质分泌。”

Tatsuya Ishihara et al.: "Cloning and characterization of the gene for a protein thiol-disulfide oxidoreductase in Bacillus brevis" Journal of Bacteriology. 177. 745-749 (1995)

Tatsuya Ishihara 等人：“短芽孢杆菌中蛋白质硫醇二硫化物氧化还原酶基因的克隆和表征”细菌学杂志。