Related Experiment Video
Updated: Jun 7, 2025
Author Spotlight: Expression and Purification of Human Solute Carrier Transporters Using Codon-Optimized Genes
Published on: September 29, 2023
Successful strategies for expression and purification of ABC transporters
Bea Berner1, Georgia Daoutsali1, Emilia Melén1
1Department and Chemistry and Molecular Biology, Gothenburg University, Box 462, 405 30 Göteborg, Sweden.
Abstract:
ATP-binding cassette (ABC) transporters are proteins responsible for active transport of various compounds, from small ions to macromolecules, across membranes. Proteins from this superfamily also pump drugs out of the cell resulting in multidrug resistance. Based on the cellular functions of ABC-transporters they are commonly associated with diseases like cancer and cystic fibrosis. To understand the molecular mechanism of this critical family of integral membrane proteins, structural characterization is a powerful tool which in turn requires successful recombinant production of stable and functional protein in good yields. In this review we have used high resolution structures of ABC transporters as a measure of successful protein production and summarized strategies for prokaryotic and eukaryotic proteins, respectively. In general, Escherichia coli is the most frequently used host for production of prokaryotic ABC transporters while human embryonic kidney 293 (HEK293) cells are the preferred host system for eukaryotic proteins. Independent of origin, at least two-steps of purification were required after solubilization in the most used detergent DDM. The purification tag was frequently cleaved off before structural characterization using cryogenic electron microscopy, or crystallization and X-ray analysis for prokaryotic proteins.
Insights
Successful recombinant production of ATP-binding cassette (ABC) transporters is crucial for understanding their mechanisms. This review summarizes strategies for producing these integral membrane proteins for structural characterization.
Area of Science:
- Biochemistry
- Structural Biology
- Molecular Biology
Background:
- ATP-binding cassette (ABC) transporters are vital membrane proteins involved in active transport.
- Dysfunctional ABC transporters are implicated in diseases such as cancer and cystic fibrosis.
- Understanding ABC transporter mechanisms requires high-resolution structural data.
Purpose of the Study:
- To review strategies for successful recombinant production of ABC transporters.
- To highlight structural characterization as a key indicator of protein production success.
- To provide insights into overcoming challenges in producing these integral membrane proteins.
Main Methods:
- Review of high-resolution structures of ABC transporters.
- Analysis of recombinant protein production strategies in prokaryotic and eukaryotic systems.
- Summary of purification techniques, including detergent solubilization and tag cleavage.
Main Results:
- Escherichia coli and HEK293 cells are common hosts for prokaryotic and eukaryotic ABC transporter production, respectively.
- At least two purification steps using DDM detergent are typically required.
- Purification tags are often removed prior to structural analysis.
Conclusions:
- Successful recombinant production is essential for structural studies of ABC transporters.
- Optimized expression and purification protocols are critical for obtaining stable, functional proteins.
- Structural characterization provides molecular insights into ABC transporter function and disease association.
Related Concept Videos
01:31ABC Transporters: Exporter
01:27ABC Transporters: Importer
In bacteria, based on the number of transmembrane helices and the chemical nature of their substrates, the ABC importers can be divided into three types: