Development of High Efficiency Protein Refolding Processes Using Molecular Chaperons.

使用分子伴侣开发高效蛋白质重折叠过程。

• 批准号: 09555238
• 负责人: FUKUDA Hideki
• 金额: $ 5.95万
• 依托单位: Kobe University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09555238/
• 关键词: Molecular chaperon; Chaperonin; GroEL; Protein refolding; Fusion protein; Affinity separation; Immobilization; 分子シャペロン; シャペロニン; GroEL

Molecular chaperons are powerful to improve the refolding yield of proteins from inclusion bodies and the reactivation of thermally inactivated enzymes in various bioprocesses. In this study, to develop the high efficiency protein refolding systems based on molecular chaperons, the properties of various types of chaperonins from different organisms (i.e. Esherichia coli, Thermu sthermophilus and Bacillus stearothermophilus) were investigated. The overexpression and purification systems of these chaperonins were constructed to produce chaperonins efficiently. To enhance the reusability of chaperonins, various immobilization methods of chaperonins were tested. As the support materials, cellulose gel beads and colloidal polymer particles were adopted. Especially, chaperonins with affinity tag (His)_6 or maltose binding protein (fusion chaperonin) were found to be effective for efficient production and immobilization using affinity adsorbents. In addition, protein refolding system using chaperonin in combination with ultrafiltration system was found to be effective. Based on these results, several efficient protein refolding systems using chaperonins were proposed.

分子伴侣在提高包涵体蛋白质的复性率和热失活酶的再活化方面具有重要作用。为了开发基于分子伴侣的高效蛋白质复性系统，本研究对来自大肠杆菌、嗜热菌和嗜热脂肪芽孢杆菌的分子伴侣蛋白的性质进行了研究。这些伴侣蛋白的过量表达和纯化系统的构建，有效地产生伴侣蛋白。为了提高伴侣蛋白的重复使用性，对伴侣蛋白的各种固定化方法进行了试验。作为载体材料，采用纤维素凝胶珠和胶体聚合物颗粒。特别是带有亲和标签（His）_6或麦芽糖结合蛋白（融合伴侣蛋白）的伴侣蛋白被发现是有效的生产和使用亲和吸附剂固定化。此外，结合超滤系统使用伴侣蛋白的蛋白质复性系统被发现是有效的。基于这些结果，提出了几种有效的蛋白质分子伴侣复性系统。

项目成果

Teshima,T.: "Effictent Protein Refolding System Using holo-Chaperonin from Thermophilic Bacterium Thermus thermophilus" Journal of Fermentation and Bioengineering. 85(6). 564-570 (1998)

Teshima,T.：“使用来自嗜热细菌Thermus thermophilus的holo-Chaperonin的有效蛋白质重折叠系统”发酵与生物工程杂志。

Teshima, T.: "Affinity Purification and Immobilization of Fusion Chaperonin GroEL-(His) 6 and Utilization to Mediate Protein Refolding" Journal of Fermentation and Bioengineering. 86 (4). 357-362 (1998)

Teshima, T.：“融合伴侣蛋白 GroEL-(His) 6 的亲和纯化和固定以及介导蛋白质重折叠的利用”发酵与生物工程杂志。

Ishii, Y.: "Single-Step Purification and Characterization of MBP (Maltose-Binding Protein)-DnaJ Fusion protein" Journal of Biochemistry. 124 (4). 842-847 (1998)

Ishii, Y.：“MBP（麦芽糖结合蛋白）-DnaJ 融合蛋白的单步纯化和表征”生物化学杂志。

Teshima,T.: "Efficient Protein Refolding System Using Holo-Chaperonin from Thermophilic Bacterium Thermus thermophilus" Journal of Fermentation and Bioengineering. 85(6). 564-570 (1998)

Teshima,T.：“使用来自嗜热细菌Thermus thermophilus的Holo-Chaperonin的高效蛋白质重折叠系统”发酵与生物工程杂志。

Teshima,T.: "Affinity Purification and Immobilization of Fusion Chaperonin GroEL-(His)_6 and Utilization to Mediate Protein Refolding" Journal of Fermentation and Bioengineering. 86(4). 357-362 (1998)

Teshima,T.：“融合伴侣蛋白 GroEL-(His)_6 的亲和纯化和固定以及介导蛋白质重折叠的利用”发酵与生物工程杂志。