Expression of active chimeric-tissue plasminogen activator in tobacco hairy roots, identification of a DNA aptamer and purification by aptamer functionalized-MWCNTs chromatography

• Published in: Protein expression and purification Vol. 152; pp. 137 - 145
• Main Authors: Nabiabad, Haidar Saify, Piri, Khosro, Amini, Massoume
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-12-2018
• Subjects: Agrobacterium - genetics; Agrobacterium - metabolism; Aptamer; Aptamers, Nucleotide - chemical synthesis; Aptamers, Nucleotide - chemistry; Chimeric-tissue plasminogen activator; Chromatography, Affinity - methods; Cloning; Cloning, Molecular; Cuscuta - genetics; Cuscuta - metabolism; Cuscutain; Enzyme Assays; Gene Expression; Genetic Vectors - chemistry; Genetic Vectors - metabolism; Hairy root; Humans; Nanotubes, Carbon - chemistry; Nicotiana - genetics; Nicotiana - metabolism; Plant Leaves - genetics; Plant Leaves - metabolism; Plant Proteins - biosynthesis; Plant Proteins - genetics; Plant Proteins - isolation & purification; Plant Roots - genetics; Plant Roots - metabolism; Plants, Genetically Modified; Protein Binding; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - isolation & purification; SELEX; SELEX Aptamer Technique; Tissue Plasminogen Activator - biosynthesis; Tissue Plasminogen Activator - genetics; Tissue Plasminogen Activator - isolation & purification; Hairy root; tPA; EDC; Cloning; SOEing-PCR; TMB; TBST; CTAB; Cuscutain; PEG; Chimeric-tissue plasminogen activator; CNTs; sulfo-NHS; Aptamer; MWCNTs; SELEX; ELISA
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2016.02.004

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 KD values of 0.320 nM and IC50 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−1 of 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.
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•Produced chimeric-tPA was showed more activity and stability.•Compared to other systems, tobacco hairy roots produced more value of tPA.•Selected aptamer possessed a good affinity toward chimeric-tPA.•Chimeric-tPA was efficiently purified by aptamer-functionalized MWCNT chromatography.