rhBMP-2m大规模制备纯化过程中使用尿素的分析

为大规模制备rhBMP-2m，将高表达rhBMP-2m的工程菌株进行高密度发酵、温度诱导表达，收集菌体，经裂菌和超声洗涤得到并纯化的包涵体。洗涤后的包涵体中，rhBMP-2mdi蛋白量的75．1％。为获得高纯度的rhBMP-2m，采用尿素作变性剂将包涵体溶解，经SP-SepharoseFF柱和Q-SepharoseFF柱两步纯化。rhBMP-2m包涵体经pH6．5尿素溶解后，经SP-Sepharose FF柱纯化，蛋白纯度为91％；若rhBMP-2m在碱性尿素中作用时间超过48h，再经Q-SepharoseFF柱纯化后，纯化产物中在还原性SDS-PAGE中会出现少量与rhBMP-2m二聚体大小接近的蛋白带，显示出碱性尿素对rhBMP-2m的共价修饰作用，会严重地影响rhBMP-2m的纯化效果和活性；若24h内完成分离纯化则影响较小，纯化后rhBMP-2m单体的纯度达98％以上。本研究表明尿素适合rhBMP-2m包涵体的大规模纯化。文章对使用尿素的利弊进行了讨论，提出大规模制备蛋白质时最好避免在碱性环境使用高浓度尿素。

Analysis of Using Urea in Large-scale Purification of rhBMP-2m Expressed from E. coli

To prepare recombinant human bone morphogenetic protein-2 mature peptide (rhBMP-2m) in large scale, the engineering strain was fermented to high density, and induced by temperature. The bacterial cells were collected and lyzed, and gained inclusive body after washing. The purity of rhBMP-2m in washed inclusive body was 75.1%. Inclusive body was dissolved in urea solution and rhBMP-2m was purified with SP-Sepharose FF and Q-Sepharose FF chromatography successively. After SP-Sepharose FF chromatography in pH6.5 buffer, the purity of rhBMP-2m was 91%. If rhBMP-2m was dissolved in alkaline urea solution over 48 h, after Q-Sepharose FF chromatography, there was a small dimer-size protein band in reductive SDS-PAGE analysis. Demonstrated that alkaline urea solution could resulted in covalent modification to rhBMP-2m, and seriously influence the purification and biological activity. If Q-Sepharose FF chromatography was completed in 24 h, the influence of alkaline urea solution was small, and the purity of rhBMP-2m was 98%. It concluded that Urea was suitable for large -scale purification of rhBMP -2m. The advantages and disadvantages of using urea were discussed, and suggested that it had better avoid using high concentration of urea in alkaline condition for large scale preperation of protein.