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Refolding of recombinant human bone morphogenetic protein-2 mature peptide in high concentration
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- Author:
- No author available
- Journal Title:
- JOURNAL OF THE FOURTH MILITARY MEDICAL UNIVERSITY
- Issue:
- 12
- DOI:
- 10.3321/j.issn:1000-2790.2008.12.005
- Key Word:
- 重组人骨形成蛋白2成熟肽;蛋白质/分离和提纯;蛋白质复性
Abstract： 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0.22 μm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.
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