Abstract
α7 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 α-conotoxin ArIB with 17-amino acid residues specifically targets α7 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 × His-tag was separately inserted in pET-32a (+) vector and transformed into Escherichia coli strain BL21(DE3) pLysS for expression. The expressions of Trx-ArIB-His6 and Trx-ArIB (V11L, V16A)-His6 were soluble in Escherichia 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 in Xenopus laevis oocytes expressing human nAChR subtypes. The results indicated that the yield of the fusion proteins was approximately 50 mg/L and rArIB (V11L, V16A) antagonized the α7 nAChR subtype selectively with 8-nM IC50. In summary, this study provides an efficient method to biosynthesize α-conotoxin ArIB and rArIB (V11L, V16A) in Escherichia coli, which could be economical to obtain massively bioactive disulfide-rich polypeptides at fast speed.
Highlights
Nicotinic acetylcholine receptors are members of ligand-gated ion channels that belong to the Cys–loop receptor superfamily [1]
The heteromeric, α9α10 nicotinic acetylcholine receptors (nAChR) subtype being expressed in outer hair cells, which is well-known for its role in the auditory system and has been shown to be involved in
Was designed and 50 -end phosphorylation synthesized, respectively using the preferred codon usage of Escherichia coli according to the mature peptide of α-CTx ArIB precursor gene (Table 1 and Figure 1), which were annealed to form double-stranded gene of rArIB and rArIB (V11L, V16A) and ligated with pET-32a(+) digested with Kpn I and EcoR I
Summary
Nicotinic acetylcholine receptors (nAChRs) are members of ligand-gated ion channels that belong to the Cys–loop receptor superfamily [1]. This superfamily includes γ-aminobutyric acidA, the 5-HT3 serotonin, and the glycine receptors [2]. NAChRs are pentameric receptors formed ligand-gated ion channels by a combination of different or same subunits. 16 subunits of nAChRs have been confirmed that are α2–10, β1–4, δ, ε and γ subunits in mammals [3,4,5], and the homomeric (α7, α8, α9 or α10) or heteromeric (α2–α6 with β2–β4, α7β2 and α9α10) receptor complexes [6,7] produces many distinctive subtypes that have different pharmacological and physiological function [8]. The heteromeric, α9α10 nAChR subtype being expressed in outer hair cells, which is well-known for its role in the auditory system and has been shown to be involved in
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