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Updated: May 10, 2026
Overexpressing and Purifying a Toxic Nuclease from Escherichia coli
Published on: August 29, 2025
Expression and secretion of functional recombinant μO-conotoxin MrVIB-His-tag in Escherichia coli
Bingmiao Gao1, Dongting Zhangsun, Yuanyan Hu
1Key Lab of Tropical Biological Resources of Ministry of Education, Key Lab for Marine Drug of Haikou, Hainan University, Hainan, Haikou 570228, China.
Abstract:
μO-conotoxin MrVIB is a 31-amino acid peptide containing three disulfide bonds isolated from the venom of Conus marmoreus, which is a selective antagonist of voltage-gated sodium channel (VGSC) Nav1.8 and has a long-lasting analgesic activity. Drug development of MrVIB has long been hindered over 15 years by difficult chemical synthesis and oxidative folding. Herein we describe a different approach based on the recombinant expression of gene MrVIB in Escherichia coli. A secretion vector pET22b(+)-MrVIB fused with pelB leader signal peptide and His-tag was constructed, which was transformed into BL21 (DE3) strain of E. coli. The recombinant conotoxin MrVIB-His-tag (rMrVIB-His) was successfully expressed and secreted into the periplasmic space of BL21 (DE3) cells. The pelB leader signal peptide was properly cleaved and three disulfide bonds were also formed properly to yield biological active rMrVIB-His. Folded rMrVIB-His in the periplasmic fraction was isolated with a Ni-NTA affinity column, which was further purified using reverse-phase high-performance liquid chromatography (RP-HPLC) and identified by liquid chromatography/mass spectrometry-ion trap-time of flight mass spectrometry (LC/MS-IT-TOF). Biological activity assay of rMrVIB-His showed it had good analgesic effects in three pain models.
Insights
Recombinant expression of μO-conotoxin MrVIB in E. coli overcomes synthesis challenges, yielding biologically active peptide for potential pain relief. This approach facilitates the development of novel analgesics targeting Nav1.8 channels.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- μO-conotoxin MrVIB, a selective Nav1.8 voltage-gated sodium channel antagonist from Conus marmoreus, exhibits potent analgesic activity.
- Traditional chemical synthesis and oxidative folding of MrVIB present significant challenges, hindering its drug development for over 15 years.
Purpose of the Study:
- To develop an alternative production method for μO-conotoxin MrVIB using recombinant DNA technology.
- To achieve efficient expression, secretion, proper folding, and purification of biologically active MrVIB in Escherichia coli.
Main Methods:
- Construction of a secretion vector (pET22b(+)-MrVIB) with a pelB leader signal peptide and His-tag for expression in E. coli BL21 (DE3).
- Isolation and purification of recombinant MrVIB-His (rMrVIB-His) from the periplasmic space using Ni-NTA affinity chromatography and RP-HPLC.
- Identification and structural confirmation of rMrVIB-His using LC/MS-IT-TOF.
Main Results:
- Successful recombinant expression and secretion of rMrVIB-His into the E. coli periplasm.
- Proper cleavage of the pelB signal peptide and formation of three disulfide bonds, resulting in biologically active rMrVIB-His.
- Purified rMrVIB-His demonstrated significant analgesic effects in preclinical pain models.
Conclusions:
- Recombinant expression in E. coli provides a viable and efficient alternative to chemical synthesis for producing μO-conotoxin MrVIB.
- This method yields correctly folded and biologically active rMrVIB-His, paving the way for its therapeutic development as a novel analgesic.
