Expression in <i>Escherichia coli</i> of fusion protein comprising α‐conotoxin Tx<scp>IB</scp> and preservation of selectivity to nicotinic acetylcholine receptors in the purified product

Yu, Jinpeng; Zhu, Xiaopeng; Yang, Yang; Luo, Sulan; Zhangsun, Dongting

doi:10.1111/cbdd.13104

Cited by 10 publications

(10 citation statements)

References 39 publications

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“…To date, over 11 conotoxins (α-TxIA, TxIB, LvIA, M-superfamily lt16a, lt15a, Bt15a, Ec15a, Cap15a, Vx15a, Vr15a, lt7a, μO-MrVIB and ω-MVIIa, PrIIIE, etc.) have successfully been recombinantly expressed in Escherichia coli [ 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 ] and bioactive TxVIA has been produced in the yeast Pichia pastoris [ 43 ].…”

Section: Discussionmentioning

confidence: 99%

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

Li¹,

Yin²,

Song³

et al. 2020

Marine Drugs

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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.

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Section: Discussionmentioning

confidence: 99%

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

Li¹,

Yin²,

Song³

et al. 2020

Marine Drugs

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“…In this context, the potential effect of a Pro-Pro motif present in the first loop of TxIA instead of the more common Pro-Ala motif might also be important due to the structural constraints induced by proline residues. However, TxIB, which appeared to fold preferentially in the globular fold (Yu et al, 2018) has a first loop with amino acid sequence identical to [R5D]TxIA, which favored the ribbon conformation in our study. Thus, factors in loop 2 of 4/7 α-conotoxins would be more likely to affect the disulfide formation.…”

Section: Discussionmentioning

confidence: 48%

“…So far, three α-conotoxins, the 4/7 α-conotoxins Vc1.1, LvIA, and TxIB, have been recombinantly expressed (Singer et al, 2012; Zhu et al, 2016; Yu et al, 2018). Vc1.1 was expressed in the non-pathogenic Salmonella enterica serovar Typhimurium strain LT2.…”

Section: Discussionmentioning

confidence: 99%

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Periplasmic Expression of 4/7 α-Conotoxin TxIA Analogs in E. coli Favors Ribbon Isomer Formation – Suggestion of a Binding Mode at the α7 nAChR

Hamdaoui

Clark

et al. 2019

Front. Pharmacol.

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Peptides derived from animal venoms provide important research tools for biochemical and pharmacological characterization of receptors, ion channels, and transporters. Some venom peptides have been developed into drugs (such as the synthetic ω-conotoxin MVIIA, ziconotide) and several are currently undergoing clinical trials for various clinical indications. Challenges in the development of peptides include their usually limited supply from natural sources, cost-intensive chemical synthesis, and potentially complicated stereoselective disulfide-bond formation in the case of disulfide-rich peptides. In particular, if extended structure–function analysis is performed or incorporation of stable isotopes for NMR studies is required, the comparatively low yields and high costs of synthesized peptides might constitute a limiting factor. Here we investigated the expression of the 4/7 α-conotoxin TxIA, a potent blocker at α3β2 and α7 nicotinic acetylcholine receptors (nAChRs), and three analogs in the form of maltose binding protein fusion proteins in Escherichia coli . Upon purification via nickel affinity chromatography and release of the toxins by protease cleavage, HPLC analysis revealed one major peak with the correct mass for all peptides. The final yield was 1–2 mg of recombinant peptide per liter of bacterial culture. Two-electrode voltage clamp analysis on oocyte-expressed nAChR subtypes demonstrated the functionality of these peptides but also revealed a 30 to 100-fold potency decrease of expressed TxIA compared to chemically synthesized TxIA. NMR spectroscopy analysis of TxIA and two of its analogs confirmed that the decreased activity was due to an alternative disulfide linkage rather than the missing C-terminal amidation, a post-translational modification that is common in α-conotoxins. All peptides preferentially formed in the ribbon conformation rather than the native globular conformation. Interestingly, in the case of the α7 nAChR, but not the α3β2 subtype, the loss of potency could be rescued by an R5D substitution. In conclusion, we demonstrate efficient expression of functional but alternatively folded ribbon TxIA variants in E. coli and provide the first structure–function analysis for a ribbon 4/7-α-conotoxin at α7 and α3β2 nAChRs. Computational analysis based on these data provide evidence for a ribbon α-conotoxin binding mode that might be exploited to design ligands with optimized selectivity.

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“…32 Several studies have attempted to produce -conotoxins recombinantly but these previous reports either did not perform structural studies or failed to produce the native "globular" isomer. 51,58,59 Using our production platform, we demonstrated the expression of [G22N]cVc1.1 in the native globular conformation. It is known that non-native -conotoxin isomers can lose potency and/or specificity.…”

Section: Scale-up Production Of Cp In Bioreactors P Pastoris Expresmentioning

confidence: 99%

An environmentally sustainable biomimetic production of cyclic disulfide-rich peptides

Yap

Looi

et al. 2020

Green Chem.

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Expression in Escherichia coli of fusion protein comprising α‐conotoxin TxIB and preservation of selectivity to nicotinic acetylcholine receptors in the purified product

Cited by 10 publications

References 39 publications

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

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

Periplasmic Expression of 4/7 α-Conotoxin TxIA Analogs in E. coli Favors Ribbon Isomer Formation – Suggestion of a Binding Mode at the α7 nAChR

An environmentally sustainable biomimetic production of cyclic disulfide-rich peptides

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