Alpha-Conotoxin As Potential To Alpha 7-Nachr Recombinant Expressed In Escherichia Coli

alpha 7 nicotinic acetylcholine receptors (nAChR) is an important nicotinic acetylcholine receptors subtype and closely associated with cognitive disorders, such as Alzheimer's and schizophrenia disease. The mutant ArIB (V11L, V16A) of alpha-conotoxin ArIB with 17-amino acid residues specifically targets alpha 7 nAChR with no obvious effect on other nAChR subtypes. In the study, the synthetic gene encoding mature peptide of ArIB and mutant ArIB (V11L, V16A) carried a fusion protein Trx and 6 x His-tag was separately inserted in pET-32a (+) vector and transformed intoEscherichia colistrain BL21(DE3) pLysS for expression. The expressions of Trx-ArIB-His(6)and Trx-ArIB (V11L, V16A)-His(6)were soluble inEscherichia coli, which were purified by Ni-NTA affinity chromatography column and cleaved by enterokinase to release rArIB and rArIB (V11L, V16A). Then, rArIB and rArIB (V11L, V16A) were purified by high-performance liquid chromatography (HPLC) and identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Bioactivity of rArIB and rArIB (V11L, V16A) was assessed by two-electrode voltage-clamp electrophysiology inXenopus laevis oocytesexpressing human nAChR subtypes. The results indicated that the yield of the fusion proteins was approximately 50 mg/L and rArIB (V11L, V16A) antagonized the alpha 7 nAChR subtype selectively with 8-nM IC50. In summary, this study provides an efficient method to biosynthesize alpha-conotoxin ArIB and rArIB (V11L, V16A) inEscherichia coli, which could be economical to obtain massively bioactive disulfide-rich polypeptides at fast speed.