Sunitha K, Chung B H, Jang K H, Song K B, Kim C H, Rhee S K
Korea Research Institute of Bioscience and Biotechnology, Taejon, Yusong, 305-600, Korea.
Protein Expr Purif. 2000 Apr;18(3):388-93. doi: 10.1006/prep.2000.1204.
Zymomonas mobilis levansucrase was overproduced by the fed-batch culture of recombinant Escherichia coli harboring a novel expression system that is constitutively expressed by the promoter from the Rahnella aquatilis levansucrase gene. Most of the levansucrase was produced as inclusion bodies in the bacterial cytoplasm, accounting for approximately 20% of the total cellular protein. Refolding after complete denaturation by high concentrations of urea or guanidine hydrochloride was not successful, resulting in large amounts of insoluble aggregates. During the development of the refolding method, it was found that direct solubilization of the inclusion bodies with Triton X-100 reactivated the enzyme, with a considerable refolding efficiency. About 65% of inclusion body levansucrase was refolded into active levansucrase in the renaturation buffer containing 4% (v/v) Triton X-100. The in vitro refolded enzyme was purified to 95% purity by single-step DEAE-Sepharose ion exchange chromatography. Triton X-100 was removed by this ion exchange chromatography.
通过对携带新型表达系统的重组大肠杆菌进行补料分批培养，使运动发酵单胞菌蔗糖转化酶过量表达，该新型表达系统由水生拉恩菌蔗糖转化酶基因的启动子组成型表达。大部分蔗糖转化酶以包涵体形式存在于细菌细胞质中，约占细胞总蛋白的20%。用高浓度尿素或盐酸胍完全变性后进行重折叠未成功，导致大量不溶性聚集体形成。在重折叠方法的开发过程中，发现用Triton X-100直接溶解包涵体可使酶重新激活，且具有相当高的重折叠效率。在含有4%（v/v）Triton X-100的复性缓冲液中，约65%的包涵体蔗糖转化酶重折叠为活性蔗糖转化酶。通过单步DEAE-琼脂糖离子交换色谱法将体外重折叠的酶纯化至95%的纯度。通过这种离子交换色谱法去除了Triton X-100。
