rhBMP-2m在高浓度条件下的复性

Refolding of recombinant human bone morphogenetic protein-2 mature peptide in high concentration

目的:优化高浓度的人骨形成蛋白2成熟肽(rh-BMP-2m)的复性条件.方法:将工程菌株进行高密度发酵、温度诱导表达,裂菌收集包涵体后经离子交换色谱分离纯化.纯化后的蛋白在不同的温度、复性液pH,氧化交换系统浓度、盐浓度等条件下进行透析复性.透析复性后进行非还原的SDS-PAGE,检测rhBMP-2m活性形式二聚体的含量,同时找出相对较优的复性方法对复性产物进行0.22μm微孔滤膜除菌并计算其损失率.结果:经透析复性后rhBMP-2m完全可溶,而且其活性形式二聚体的含量达到30%左右,即复性后二聚体的rhBMP-2m可以达到每升发酵液800 mg,并且经微孔滤膜除菌后rhBMP-2m活性形式二聚体的含量基本没有损失,同时蛋白定量也表明过滤除菌后蛋白损失不超过10%.结论:高pH有利于rhBMP-2m的复性,除变性剂尿素时采用低pH可以保持高浓度rhBMP-2m复性后仍保持可溶.蛋白质的浓度、pH和温度对蛋白复性的结果影响很大,但盐浓度、氧化交换系统的浓度和透析外液体积对蛋白复性影响不是很大.

AIM： To optimize the refolding of recombinant human bone morphogenetie protein-2 mature peptide （rhBMP-2m） in high protein concentration. METHODS： The engineered strain, which could express rhBMP-2m at a high level, was fermented in high density, and induced by temperature. The bacterial cells were collected and analyzed. The gained inclusion bodies were purified by ion-exchange chromatography. The purified rh- BMP-2m was refolded by different dialysis methods, especially changing the pH, temperature, oxidize-reduced pair and ion concentration. After that, the refolding results were analyzed by nonreducing SDS-PAGE. The refolding rhBMP-2m passed through a 0.22 μm micropore filter membrane for degermation, and then the loss percentage was analyzed. RESULTS： The refolding rhBMP- 2m was completely soluble, and more than 30% of rhBMP-2m dimmer were obtained corresponding to 800 mg rhBMP-2m dimmer per liter of culture broth, and the dimmer percent of refolding rhBMP-2m was remained after degerming by micropore filter membrane. At the same time, protein quantitative analysis showed that the loss of refolding rhBMP-2m was no more than 100 g/L. CONCLUSION： Changing pH during the refolding process can make the refolding production completely soluble, and the high pH is beneficial for refolding and low pH keeps the refolding rhBMP-2m soluble even if removing the deforming agent Urea from the high concentration of the refolding protein. The concentration of the protein, pH and the temperature affect the refolding significantly, while the concentration of ion and oxidize-reduced pair, and volume of dialysis refolding buffer have little effect.