Related Experiment Video
Updated: Nov 8, 2025
10:28
The Encapsulation of Cell-free Transcription and Translation Machinery in Vesicles for the Construction of Cellular Mimics
Published on: October 21, 2013
15.4K
Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles
Jasmine M Hershewe1,2,3, Katherine F Warfel1,2,3, Shaelyn M Iyer1
1Department of Chemical and Biological Engineering, Northwestern University, Technological Institute E136, Evanston, IL, 60208, USA.
Nature Communications
|April 23, 2021
Summary
This study enhances cell-free gene expression (CFE) systems by enriching native membrane vesicles. These modified CFE systems improve the production of complex glycoproteins through bacterial glycoengineering.
Area of Science:
- Synthetic Biology
- Biotechnology
- Molecular Biology
Background:
- Cell-free gene expression (CFE) systems are valuable for biomanufacturing and synthetic biology.
- A key limitation in bacterial CFE systems is the activation of membrane-dependent functions using cell-derived vesicles.
Purpose of the Study:
- To characterize native membrane vesicles in Escherichia coli CFE extracts.
- To develop methods for enriching these vesicles with heterologous membrane proteins for enhanced functionality.
- To apply these methods to bacterial glycoengineering for improved glycoprotein synthesis.
Main Methods:
- Characterization of native membrane vesicles using orthogonal techniques.
- Optimization of extract processing to increase membrane vesicle concentration.
- Enrichment of vesicles with heterologous membrane proteins, specifically oligosaccharyltransferases.
- Application to cell-free synthesis of N-linked and O-linked glycoproteins.
Main Results:
- Established methods to increase membrane vesicle concentration in CFE extracts.
- Demonstrated enhanced concentrations of heterologous membrane protein cargo in enriched extracts.
- Successfully improved cell-free glycoprotein synthesis by enriching membrane-bound enzymes and substrates.
Conclusions:
- The developed methods facilitate the enrichment of membrane vesicles in CFE systems.
- This approach enhances membrane-associated activities, particularly for bacterial glycoengineering.
- Enables on-demand glycoprotein production and opens avenues for novel CFE systems with membrane functions.
