Efficient expression and biological activity characterization of human potassium channel KV3.1 in an Escherichia coli cell-free protein synthesis system.
- Author:
Zitong ZHAO
1
;
Tianqi ZHOU
1
;
Yunyang SONG
1
;
Fanghui WU
1
;
Yifeng YIN
1
;
Yanli LIU
2
,
3
Author Information
1. State Key Laboratory of NBC Protection for Civilian, Beijing 102205, China.
2. State Key Laboratory of NBC Protection for Civilian, Beijing 102205, China. *Corresponding author, E-mail: liuh3062023@
3. com.
- Publication Type:Journal Article
- MeSH:
Humans;
Escherichia coli/metabolism*;
Shaw Potassium Channels/biosynthesis*;
Cell-Free System;
Circular Dichroism;
Protein Biosynthesis;
Recombinant Proteins/metabolism*;
Membrane Potentials;
Shab Potassium Channels
- From:
Chinese Journal of Cellular and Molecular Immunology
2025;41(11):1000-1006
- CountryChina
- Language:Chinese
-
Abstract:
Objective This study aims to achieve high-yield functional expression of the human voltage-gated potassium channel KV3.1 using an Escherichia coli cell-free protein synthesis system, thereby providing a novel synthetic approach for drug screening, structural analysis and functional characterization of KV3.1. Methods KV3.1 was expressed in an Escherichia coli cell-free protein synthesis system for 10 hours in the presence of peptide surfactant A6K. The secondary structure of KV3.1 was analyzed by circular dichroism spectroscopy. The potassium channel activity of the recombinant protein liposome KV3.1-A6K was investigated using fluorescent dyes Oxonol VI as indicators, which are capable of reflecting alterations in membrane potential. Results Soluble KV3.1 protein was successfully synthesized, achieving a purified yield of up to 1.2 mg/mL via an Escherichia coli cell-free protein synthesis system. Circular dichroism spectroscopy revealed that KV3.1 exhibited characteristic α-helical secondary structures. Membrane potential fluorescence assays demonstrated that the KV3.1-A6K proteoliposomes, which were reconstructed with surfactant peptide A6K, exhibited remarkable potassium ion permeability. Conclusion This study successfully achieved high-yield expression of human KV3.1 with activity using an Escherichia coli-based cell-free protein synthesis system. This innovative method not only significantly enhances the expression yield of KV3.1, but also maintains its functional activity, thereby establishing a novel and efficient synthetic platform for drug screening and advancing our understanding of structure-function relationships in KV3.1 research.
