Expression vector for recombinant u-conotoxin THIA or tiiialamut expression in e.coli

The novel expression vector construct for recombinant p-conotoxin TIIIA or TIIIAlaMut in E. coli addresses the inefficiencies of existing methods by enabling continuous protein synthesis and controlled disulfide bridge formation, resulting in efficient and cost-effective production of p-conotoxins.

WO2025057018A1 Publication Date: 2025-03-20SCIENCE4BEAUTY SP ZOO
0 Cites 0 Cited by

Patent Information

Application Number
PCT/IB2024/058584
Authority / Receiving Office
WO · WO
Patent Type
Applications
Current Assignee / Owner
Priority Date
2023-09-05
Filing Date
2024-09-04
Publication Date
2025-03-20

AI Technical Summary

Technical Problem

Current methods for producing p-conotoxins, such as chemical synthesis and extraction from sea snails, are costly and inefficient, while bacterial expression systems face challenges with post-translational modifications and abnormal folding.

Method used

A novel expression vector construct for recombinant p-conotoxin TIIIA or TIIIAlaMut expression in E. coli, featuring a TRX::TIIIA or TRX::TIIIAlaMut fusion protein with a leader protein modified by point mutagenesis, and a constitutive deoP1P2 promoter, enabling continuous protein synthesis without IPTG induction.

Benefits of technology

This method allows for the efficient production of soluble p-conotoxin in E. coli, with controlled disulfide bridge formation ex vivo, reducing costs and improving yield compared to traditional methods.

✦ Generated by Eureka AI based on patent content.
Patent Text Reader

Abstract

Subject matter of the invention is a construct for an expression vector for recombinant μ-conotoxin TIIIA or TIIIAlaMut expression, characterized in that it contains either the nucleotide sequence of μ-conotoxin TIIIA SEQ ID NO: 1 or the nucleotide sequence of μ-conotoxin TIIIAlaMut SEQ ID NO: 4, both of which at the 5' end contain a sequence encoding six histidines (6His) linked by a serine-glycine-serine linker (SGS) to a construct encoding a TRX::TIIIA fusion protein containing the μ-conotoxin TIIIA gene and the gene encoding a leader protein, or a TRX::TIIIAlaMut fusion protein containing the μ-conotoxin TIIIAlaMut gene and a leader protein, where the said leader protein is thioredoxin (TRX) modified by point mutagenesis in which the amino acid methionine at position 37 has been replaced by lysine. Another subject matter of the invention is an expression vector containing the construct according to the invention under the control of a constitutive promoter. Another subject matter of the invention is an isolated E. coli cell containing the expression vector according to the invention. Another subject matter of the invention is a method for producing μ-conotoxin TIIIA or TIIIAlaMut in E. coli with the expression vector containing the construct according to the invention, characterized in that it includes the following steps: a) Transformation of an E. coli cell with the expression vector containing the construct according to the invention, under the control of a constitutive promoter, encoding either a TRX::TIIIA fusion protein with amino acid sequence SEQ ID NO: 2 or a TRX::TIIIAlaMut fusion protein with SEQ ID NO: 5; b) Expression of the TRX::TIIIA or TRX::TIIIAlaMut fusion protein; c) Isolation and purification of the TRX::TIIIA or TRX::TIIIAlaMut fusion protein; d) Disulfide bridges forming by subjecting the purified TRX::TIIIA or TRX:: TIIIAlaMut fusion protein to glutathione treatment in GSH / GSSG and dialysis in buffer; e) Cyanogen bromide cleavage of the TRX::TIIIA or TRX::TIIIAlaMut fusion protein with disulfide bridges formed; f) Purification of the cleaved TIIIA or TIIIAlaMut peptide.
Need to check novelty before this filing date? Find Prior Art