Nematollahi Leila, Khalaj Vahid, Babazadeh Seyedeh Maliheh, Rahimpour Azam, Jahandar Hoda, Davami Fatemeh, Mahboudi Fereidoun
Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
Avicenna J Med Biotechnol. 2012 Oct;4(4):178-85.
Bone Morphogenetic Proteins (BMPs) belong to the transforming growth factor-β (TGF-β) superfamily, and play an important role in bone metabolism. Recombinant forms of BMP-2 and BMP-7 are the only BMPs used clinically. In this study the mature part of human bone morphogenetic protein-7 (BMP-7) was engineered through substitution of the BMP-7 N-terminal sequence by heparin-binding site of BMP-2. This targeted substitution was made to enhance the binding affinity of the novel protein to the extracellular matrix components such as heparin and heparan sulfate proteoglycans (HSPGs).
The engineered protein was expressed in Escherichia coli (E.coli). The PelB signal sequence was used to translocate soluble proteins into the periplasmic space of E.coli. The protein was purified from periplasmic extract using Ni-NTA chromatography and the SDS-PAGE and western blot analysis confirmed the successful expression of the novel protein.
The novel hBMP-7 mutant was produced as approximately 16 kDa monomer. It was found that the heparin binding of this protein was approximately 50% more than that of the wild-type at a protein concentration of 500 ng/ml.
The findings showed that the periplasmic expression may be suitable to produce complex proteins like BMPs.
骨形态发生蛋白（BMPs）属于转化生长因子-β（TGF-β）超家族，在骨代谢中起重要作用。重组形式的BMP-2和BMP-7是临床上唯一使用的BMPs。在本研究中，通过用BMP-2的肝素结合位点替换BMP-7的N端序列，对人骨形态发生蛋白-7（BMP-7）的成熟部分进行了工程改造。进行这种靶向替换是为了增强新蛋白与细胞外基质成分如肝素和硫酸乙酰肝素蛋白聚糖（HSPGs）的结合亲和力。
工程蛋白在大肠杆菌（E.coli）中表达。PelB信号序列用于将可溶性蛋白转运到大肠杆菌的周质空间。使用Ni-NTA色谱从周质提取物中纯化蛋白，SDS-PAGE和western blot分析证实了新蛋白的成功表达。
新的hBMP-7突变体以约16 kDa的单体形式产生。发现在蛋白浓度为500 ng/ml时，该蛋白的肝素结合能力比野生型高约50%。
研究结果表明，周质表达可能适合生产像BMPs这样的复杂蛋白。
