Development of High Efficiency Protein Refolding Processes Using Molecular Chaperons.

分子伴侣在提高包涵体中蛋白质的复性产率和提高热失活的酶在各种生物过程中的活性方面具有很强的作用。为了开发基于分子伴侣的高效蛋白质复性体系，本研究对来自不同生物(即大肠杆菌、嗜热嗜热杆菌和嗜热脂肪芽孢杆菌)的不同类型的伴侣蛋白的性质进行了研究。构建了这些伴侣蛋白的高效表达和纯化系统，以高效生产伴侣蛋白。为了提高分子伴侣的可重用性，对各种分子伴侣的固定化方法进行了试验。载体材料采用纤维素凝胶小球和胶体聚合物颗粒。尤其是带有亲和标签(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利用亲和吸附剂高效生产和固定化的有效方法。此外，将伴侣蛋白与超滤系统相结合的蛋白质复性系统也是有效的。在此基础上，提出了几种高效的伴侣蛋白复性体系。

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.