title: Expression of active chimeric-tissue plasminogen activator in tobacco hairy roots, identification of a DNA aptamer and purification by aptamer functionalized-MWCNTs chromatography.
abstract: 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 KDvalues of 0.320 nM and IC50of 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-1of total protein, respectively. The activities of chimeric-tPA and normal-tPA were 90 and 60 IUml-1, respectively. Compared to the normal-tPA, chimeric-tPA showed more activity.
authors.affiliation: Department of Biotechnology, College of Agriculture, Bu-Ali Sina University, Hamadan, Iran.
authors.forename: Haidar Saify
authors.initials: HS
authors.lastname: Nabiabad
authors.affiliation: Department of Biotechnology, College of Agriculture, Bu-Ali Sina University, Hamadan, Iran. Electronic address: kh_piri@basu.ac.ir.
authors.forename: Khosro
authors.initials: K
authors.lastname: Piri
authors.affiliation: Department of Biotechnology, College of Agriculture, Bu-Ali Sina University, Hamadan, Iran.
authors.forename: Massoume
authors.initials: M
authors.lastname: Amini
mesh_terms: D060054:Agrobacterium / Q000235:genetics / Q000378:metabolism; D052157:Aptamers, Nucleotide / Q000138:chemical synthesis / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D003001:Cloning, Molecular; D034182:Cuscuta / Q000235:genetics* / Q000378:metabolism; D057075:Enzyme Assays; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D037742:Nanotubes, Carbon / Q000737:chemistry; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D018517:Plant Roots / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D052156:SELEX Aptamer Technique; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism
chemical_list: D052157:Aptamers, Nucleotide; D037742:Nanotubes, Carbon; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D010959:Tissue Plasminogen Activator
keywords: Aptamer; Chimeric-tissue plasminogen activator; Cloning; Cuscutain; Hairy root; SELEX