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 biosurfaces so can be used as a topical therapeutic agent without injection. To maintain the functionality of TAT-BoNT/A , 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.KEYWORDS cell-penetrating peptide; inclusion body; light chain of botulinum toxin type A; protein expression TAT-BoNT/A

Expression and Purification of Extracellular Solute-Binding Protein (ESBP) in Escherichia coli, the Extracellular Protein Derived from Bifidobacterium longum KACC 91563

“…We could produce the recombinant protein under native condition, while no visible protein band of the appropriate molecular weights was observed with denaturing condition. Purification under native conditions is an ideal condition to preserve its biological activity and avoid significant loss of produced protein during the renaturation step (Saffarian et al, 2016). After the parameters affecting the amount of purified protein were explored and optimized, total of 7.15 mg of ESBP were produced from two liters of transformed culture.…”

The Expanding Therapeutic Utility of Botulinum Neurotoxins

“…Perhaps the most unusual and potentially risky uses of CPP to achieve transdermal delivery of BoNT was the direct fusion of the TAT peptide to the light chain of BoNT/A [ 160 , 161 ]. This was reported to enable the direct application and delivery of the light chain into cells without the need for any of the other domains of BoNT that mediate neuronal specific binding and the translocation of the light chain into the cytosol [ 160 ].…”