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首页> 外文期刊>Molecular Biology Reports >Optimized procedure for expression and renaturation of recombinant human bone morphogenetic protein-2 at high protein concentrations
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Optimized procedure for expression and renaturation of recombinant human bone morphogenetic protein-2 at high protein concentrations

机译：高蛋白浓度下重组人骨形态发生蛋白2表达和复性的优化程序

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摘要

A prokaryotic expression system has been used to produce recombinant human bone morphogenetic protein-2 (rhBMP-2). However, low rhBMP-2 yields and protein loss during purification and renaturation are the hurdles in the clinical application. Previous studies have indicated that variables such as temperature, host cell, salt concentration, and culture time affect the final rhBMP-2 yield. The optimization of these conditions in an Escherichia coli culture yielded 28.258 mg of rhBMP-2 per liter of culture. To reduce rhBMP-2 loss during purification and renaturation, we performed purification before renaturation in the prokaryotic expression system instead of using the traditional renaturation-before-purification approach. rhBMP-2 was separated on a Sephacryl S-300 HR column and eluted from a DEAE-Sepharose Fast Flow column. The collected protein was refolded by dialysis with urea buffer, which was followed by dialysis with ultrapure water. The purified rhBMP-2 dimer significantly increased alkaline phosphatase (ALP) activity and osteogenic activity in the femoral muscle and showed the same level of bone-forming activity as natural BMP-2. This optimized procedure for expression and renaturation of rhBMP-2 has potential clinical applications.
机译：原核表达系统已用于生产重组人骨形态发生蛋白2（rhBMP-2）。然而，在纯化和复性过程中，低的rhBMP-2产量和蛋白质损失是临床应用的障碍。先前的研究表明，诸如温度，宿主细胞，盐浓度和培养时间等变量会影响最终的rhBMP-2产量。在大肠杆菌培养物中优化这些条件可得到每升培养物中28.258 mg rhBMP-2。为了减少纯化和复性过程中的rhBMP-2损失，我们在原核表达系统中复性前进行了纯化，而不是使用传统的复性前-纯化方法。在Sephacryl S-300 HR色谱柱上分离rhBMP-2，并从DEAE-Sepharose Fast Flow色谱柱上洗脱。通过用尿素缓冲液透析来重新折叠收集的蛋白质，然后用​​超纯水进行透析。纯化的rhBMP-2二聚体显着增加了碱性磷酸酶（ALP）活性和股骨中成骨活性，并显示了与天然BMP-2相同水平的骨形成活性。这种用于rhBMP-2表达和复性的优化程序具有潜在的临床应用。

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