从重组融合蛋白包涵体中释放大环肽过程优化

Process optimization of dissolving recombinant cyclotide-fused protein inclusion bodies forre leasing cyclotide

[目的]探索不同条件对包涵体复性的影响,优化重组融合蛋白包涵体中释放大环肽的工艺条件。[方法]对纯化后的包涵体进行不同变性液、透析方式及是否超声破碎处理的比较;并在此基础上探讨不同透析温度、谷胱甘肽浓度与L-Arg浓度对大环肽产量的影响。采用Tanon凝胶分析系统与紫外吸收法对大环肽的产量进行分析。[结果]使用8 mol/L的尿素溶解包涵体后,获得的上清液在25℃条件下,在含有1 mmol/L GSH、0.1 mmol/L GSSG、0.8mmol/L L-Arg的透析液中进行梯度透析,其得率为每克包涵体获得约1.4 mg大环肽。[结论]成功从包涵体中获得大环肽并优化工艺条件。

[ Objective ] This study aims to optimize the conditions of a kind of cyclic peptides, cyclotides, released from their fused cyclotide - intein - CBD （ chitin - binding domain） proteins by exploring the effects of different conditions. [ Methods ] Firstly, purified inclusion bodies were treated with or without ultrasonication while it dissolved by 6 mol/L guanidine HC1 or 8 mol/L urea. Secondly, dialyzed the dissolved proteins in the dialysis solution of which with gradually decreasing concentrations of urea under different temperatures. Thirdly, on the basis of the above conditions, dialyzed the dissolved proteins in the dialysis solution of which containing different concentrations of GSH/GSSG and L - Arg. Finally, evaluated releasing conditions of cyclotides from their fused protein inclusion bodies using gel - electrophoresis, Tanon gel analysis system and ultraviolet absorption. [ Results] On the basis of above noted experiment resuhs, a suite of technological conditions were proposed as below. The inclusion bodies were dissolved by 8mol/L urea, and the dissolved proteins were dialyzed in renaturing solution containing 1 mmol/L GSH,0.1 mmol/L GSSG,and 0. 8 mmol/L L - Arg at 25 ℃. The finally reduction rate of cyclotides is 1.4 mg from 1 g inclusion bodies. [ Conclusion ] The above proposed technological processes can be used for producing those recombinant peptides,which need to be released from their fused proteins with intein - CBD that are expressed in the forms of inclusion bodies.