α-Conotoxin as Potential to α7-nAChR Recombinant Expressed in Escherichia coli

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.

Usage of Cell-Free Protein Synthesis in Post-Translational Modification of μ-Conopeptide PIIIA

“…This confirmed the effectiveness of the process in CE-CFPS, where P4H catalyzed the hydroxylation of proline through the recognition of the pro region of the precursor µ-PIIIA. Hence, in contrast to the yield of ArIB in our earlier study [49], the production of fusion rPIIIA in Escherichia coli was much lower, at approximately 20 mg/L in flasks, which might be attributed to the co-expression of the hybrid precursors of µ-PIIIA and P4H, even though they did not share a common T7 promoter. The post-translational modifications have an important effect on the structure and bioactivity of conopeptides.…”

Marine natural products

“…Successful recombinant expression of the α7-nAChR antagonist α-conotoxin ArIB and a double residue mutant in Escherichia coli has been reported. 666 Molecular dynamics simulations have been used to study the interaction of α-conotoxin LsIA and a C-terminus carboxylated analogue with α3β2 nAChR, 667 while antagonists α-conotoxin TxID and a single residue mutant have been used to determine that α3b4 nAChR may be a potential target for anti-nicotine addiction treatment. 668 Functional investigation of αM-conotoxin MIIIJ ( C. magus ) has revealed it to be an inhibitor of muscular nAChRs by interfering with acetylcholine binding.…”

Strategies for Heterologous Expression, Synthesis, and Purification of Animal Venom Toxins

“…To date, bacterial expression still remains the preferred system for the heterologous expression of toxins, especially for small and cysteine-less toxins like actinoporins from sea anemones ( Alegre-Cebollada et al, 2007 ). Bacterial expression has been successfully used to produce the majority of scorpion toxins produced so far ( Amorim et al, 2018 ), and it has been widely used to express snake toxins ( Clement et al, 2016 ; Shulepko et al, 2017 ; David et al, 2018 ; Guerrero-Garzón et al, 2018 ; Russo et al, 2019 ), conotoxins from cone snails ( Yu et al, 2018 ; Nielsen et al, 2019 ; Liu et al, 2020 ), and spider toxins ( Meng et al, 2011 ; Souza et al, 2012 ; Chassagnon et al, 2017 ; Wu et al, 2017 ). Nevertheless, as mentioned before, PTMs and notably, complex disulfide-bonding patterns pose a considerable challenge for the general use of bacterial expression systems.…”

“…In the last years, several other noteworthy toxin-derived drugs with novel medical applications have entered clinical trials ( Bordon et al, 2020 ). Dalazatide, a synthetic peptide derivative of a toxin from the sun sea anemone ( Stichodactyla helianthus ), which toxicity relies on inhibiting voltage-gated potassium channel Kv1.3, has demonstrated efficacy in the treatment of autoimmune disorders, including psoriasis, arthritis, multiple sclerosis, lupus, and rheumatoid arthritis ( Liu et al, 2020 ). Desmoteplase, a recombinant toxin derivative from vampire bat venom, with a function similar to tissue plasminogen activator, has applications in acute ischemic stroke ( Reddrop et al, 2005 ).…”