Nabiabad Haidar Saify, Piri Khosro, Amini Massoume
Department of Biotechnology, College of Agriculture, Bu-Ali Sina University, Hamadan, Iran.
Department of Biotechnology, College of Agriculture, Bu-Ali Sina University, Hamadan, Iran.
Protein Expr Purif. 2018 Dec;152:137-145. doi: 10.1016/j.pep.2016.02.004. Epub 2016 Feb 12.
Tissue-type plasminogen activator (tPA) is a serine protease that plays a crucial role in the fibrinolytic system. We increased the activity of tPA by splicing the active site of dodder-cuscutain gene to human tPA. The chimeric cDNA of tPA was constructed by Splicing by Overlap Extension Polymerase Chain Reaction (SOEing-PCR) method and transferred to the hairy roots of tobacco using different strains of Agrobacterium rhizogenes. Chimeric-tPA was purified by lysine-sepharose chromatography and specific aptamers were designed using SELEX method. Multi wall carbon nanotubes were functionalized with selected aptamers, packed in a column, and used for purification. The results demonstrated that selected aptamer having K values of 0.320 nM and IC of 28.9 nM possessed good affinity to tPA, and the chimeric-tPA was properly purified by aptamer-chromatography. Hairy roots expressing chimeric-tPA and normal-tPA produced 900 and 450 ngmg of total protein, respectively. The activities of chimeric-tPA and normal-tPA were 90 and 60 IUml, respectively. Compared to the normal-tPA, chimeric-tPA showed more activity.
组织型纤溶酶原激活剂（tPA）是一种丝氨酸蛋白酶，在纤维蛋白溶解系统中起关键作用。我们通过将菟丝子菟丝素基因的活性位点与人类tPA拼接来提高tPA的活性。tPA的嵌合cDNA通过重叠延伸聚合酶链反应（SOEing-PCR）方法构建，并使用不同菌株的发根农杆菌转移到烟草毛状根中。嵌合tPA通过赖氨酸-琼脂糖层析纯化，并使用SELEX方法设计特异性适体。多壁碳纳米管用选定的适体进行功能化，填充在柱中并用于纯化。结果表明，K值为0.320 nM且IC为28.9 nM的选定适体对tPA具有良好的亲和力，并且嵌合tPA通过适体层析得到了恰当的纯化。表达嵌合tPA和正常tPA的毛状根分别产生了900和450 ng/mg的总蛋白。嵌合tPA和正常tPA的活性分别为90和60 IU/ml。与正常tPA相比，嵌合tPA表现出更高的活性。
