title: TAT-BoNT/A(₁₋₄₄₈), a novel fusion protein as a therapeutic agent: analysis of transcutaneous delivery and enzyme activity.
abstract: Botulinum neurotoxin type A (BoNT/A) has been used as an injectable therapeutic agent for the treatment of some abnormal muscle contractions. In this study, TAT(47-57) peptide, a cell-penetrating peptide, was fused with the catalytic domain of BoNT/A for therapeutic purposes. HeLa and BE(2)-C cell lines were treated separately with purified TAT-BoNT/A(1-448) recombinant protein, and transduction of protein was analyzed by western blotting. Also, transcutaneous delivery through mouse skin surface was evaluated by immunohistochemistry. The in vitro catalytic activity of TAT-BoNT/A(1-448) was evaluated by HPLC. The presence of recombinant protein was detected in both of the cell lines as well as mouse skin cryosections after 60 and 120 min of incubation. The concentration of intracellular proteins was increased over time. HPLC analysis showed that this fusion protein has a biological activity 1.5 times as much as the full-length BoNT/A(1-448) protein. TAT-BoNT/A(1-448) fusion protein is biologically active and can transmit through living cells in vitro and in vivo successfully and more effectively compared with BoNT/A(1-448) protein as control.
authors.affiliation: Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
authors.forename: Parvaneh
authors.initials: P
authors.lastname: Saffarian
authors.affiliation: Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran. najarp_s@modares.ac.ir.
authors.forename: Shahin Najar
authors.initials: SN
authors.lastname: Peerayeh
authors.affiliation: Applied Microbiology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran.
authors.forename: Jafar
authors.initials: J
authors.lastname: Amani
authors.affiliation: Department of Biology, Faculty of Basic Sciences, Imam Hussein University, Tehran, Iran.
authors.forename: Firooz
authors.initials: F
authors.lastname: Ebrahimi
authors.affiliation: Applied Microbiology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran.
authors.forename: Hamid
authors.initials: H
authors.lastname: Sedighian
authors.affiliation: Applied Microbiology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran.
authors.forename: Raheleh
authors.initials: R
authors.lastname: Halabian
authors.affiliation: Applied Microbiology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran. imanifouladi.a@gmail.com.
authors.forename: Abbas Ali Imani
authors.initials: AA
authors.lastname: Fooladi
mesh_terms: D000279:Administration, Cutaneous; D000818:Animals; D000927:Anticonvulsants / Q000008:administration & dosage / Q000378:metabolism* / Q000493:pharmacokinetics; D015153:Blotting, Western; D019274:Botulinum Toxins, Type A / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism* / Q000493:pharmacokinetics; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism* / Q000493:pharmacokinetics
chemical_list: D000927:Anticonvulsants; D011993:Recombinant Fusion Proteins; D019274:Botulinum Toxins, Type A
keywords: Botulinum neurotoxin type A; Cell transduction; Fusion protein; Protein purification; TAT peptide; Transcutaneous delivery