Khodabakhsh Fatemeh, Dehghani Zohreh, Zia Mohammad Farid, Rabbani Mohammad, Sadeghi Hamid Mir Mohammad
Department of Pharmaceutical Biotechnology, Isfahan Pharmaceutical Sciences Research Centre, School of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran.
Avicenna J Med Biotechnol. 2013 Jul;5(3):168-75.
Production of tissue Plasminogen Activator protein (t-PA) in prokaryotes systems has many problems such as the lack of active protein production, multiple purification steps, and renaturation process which has been shown to be costly and time-consuming.
In this study, reteplase which is the nonglycosylated active domain of t-PA was used to transform TOP10 Escherichia coli (E. coli) bacteria to resolve some of the above mentioned problems. Reteplase cDNA was ligated into pBAD/gIII plasmid which allowed secretion of this protein into the periplasmic space and would allow the correct formation of disulfide bonds in protein structure. The presence of reteplase cDNA in pBAD/gIII plasmid was confirmed by restriction digestion and sequencing. After induction of the expression of this protein by adding 0.0002% L-Arabinose to the medium, the proteins in periplasmic space as well as the inclusion bodies formed inside the cell were extracted. Subsequently, these proteins were purified and detected by Western blot method.
Our results showed that the amount of reteplase extracted from periplasmic space was much lower than the extracted inclusion bodies and large quantities of the recombinant protein were present as inclusion bodies. Therefore, it was more efficient to use inclusion body extraction method for protein isolation and purification.
We produced active reteplase after its expression in E. coli TOP10 and isolation of inclusion bodies produced the best results for purification and extraction of this protein.
在原核生物系统中生产组织型纤溶酶原激活物蛋白（t-PA）存在诸多问题，如缺乏活性蛋白产生、多个纯化步骤以及复性过程，而复性过程已被证明成本高且耗时。
在本研究中，使用t-PA的非糖基化活性结构域瑞替普酶来转化TOP10大肠杆菌（E. coli）细菌，以解决上述一些问题。将瑞替普酶cDNA连接到pBAD/gIII质粒中，该质粒可使该蛋白分泌到周质空间，并能使蛋白结构中正确形成二硫键。通过限制性酶切和测序确认pBAD/gIII质粒中存在瑞替普酶cDNA。向培养基中添加0.0002% L-阿拉伯糖诱导该蛋白表达后，提取周质空间中的蛋白以及细胞内形成的包涵体。随后，对这些蛋白进行纯化并通过蛋白质印迹法进行检测。
我们的结果表明，从周质空间提取的瑞替普酶量远低于提取的包涵体，并且大量重组蛋白以包涵体形式存在。因此，使用包涵体提取方法进行蛋白分离和纯化效率更高。
我们在大肠杆菌TOP10中表达瑞替普酶后获得了活性瑞替普酶，分离包涵体对该蛋白的纯化和提取效果最佳。
