Expression and refolding of bioactive α-bungarotoxin V31 in E. coli

•Optimized coding sequences produced rαBtx in E. coli without fusion tags.•Optimized simple protocol produced rαBtx similar to its native counterpart.•Ultrasound used in cell lysis can be detrimental to inclusion bodies if overdosed.

In order to obtain bioactive α-bungarotoxin (αBtx) using recombinant protein technique, a codon-optimized synthetic gene was expressed in fusion with the N-terminal 10-His-tag and C-terminal Strep-tag in Escherichia coli. Further optimization through site-directed mutagenesis enabled moderate expression of the protein without the N-terminal His-tag or the C-terminal Strep-tag. Two such recombinant αBtx (rαBtx) were obtained, both with an additional methionine and a glycine at the N-terminal and one with (G4S1)2-Strep-tag at the C-terminal. The rαBtx proteins were refolded using a novel protocol, which efficiently produced final products with activity similar to its natural counterpart. The protocol could easily be scale up, which produced 0.3–1 mg of pure and highly active rαBtx per liter of E. coli culture.