title: Expression and purification of recombinant TAT-BoNT/A(1-448)under denaturing and native conditions.
abstract: Botulinum toxin type A can temporarily inhibit muscle contraction. Currently, physicians administer this toxin as a bio-drug in treatment of some muscle contraction disorders. TAT-BoNT/A(1-448)is a functional recombinant protein derived from botulinum toxin light chain. Unlike the full length botulinum toxin, TAT-BoNT/A(1-448)is a self-permeable molecule which can pass through bio-surfaces so can be used as a topical therapeutic agent without injection. To maintain the functionality of TAT-BoNT/A(1-448), it is necessary to restore its normal folding upon expression and purification. In this study, we have investigated and optimized expression conditions for this novel recombinant protein. Under denaturing condition (1 mM IPTG, at 37°C), the chimeric protein was produced as inclusion body and required to be purified using denaturing agents (e.g. urea). Yet, lower incubation temperature (18°C) and less IPTG concentration (0.5 mM) induce a protein under native condition. In such condition, about 60% of the chimeric protein was expressed in soluble form.
authors: a Department of Microbiology , Tehran Medical Sciences Branch, Islamic Azad University , Tehran , Iran.
authors: Parvaneh
authors: P
authors: Saffarian
authors: b Department of Bacteriology , Faculty of Medical Sciences, Tarbiat Modares University , Tehran , Iran.
authors: Shahin Najar
authors: SN
authors: Peerayeh
authors: d Department of Biology , Faculty of Basic Sciences, Imam Hussein University , Tehran , Iran.
authors: Jafar
authors: J
authors: Amani
authors: c Applied Microbiology Research Center, Baqiyatallah University of Medical Sciences , Tehran , Iran.
authors: Firooz
authors: F
authors: Ebrahimi
authors: c Applied Microbiology Research Center, Baqiyatallah University of Medical Sciences , Tehran , Iran.
authors: Hamid
authors: H
authors: Sedighianrad
authors: c Applied Microbiology Research Center, Baqiyatallah University of Medical Sciences , Tehran , Iran.
authors: Raheleh
authors: R
authors: Halabian
authors: c Applied Microbiology Research Center, Baqiyatallah University of Medical Sciences , Tehran , Iran.
authors: Abbas Ali
authors: AA
authors: Imani Fooladi
mesh_terms: D019274:Botulinum Toxins, Type A / Q000096:biosynthesis* / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D007218:Industrial Microbiology / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D019274:Botulinum Toxins, Type A
keywords: TAT-BoNT/A(1–448); cell-penetrating peptide; inclusion body; light chain of botulinum toxin type A; protein expression