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A Flexible and Scalable High-Throughput Platform for Recombinant Membrane Protein Production
Hui Xu1, Thomas Clairfeuille1, Christine C Jao1
1Department of Structural Biology, Genentech Inc., South San Francisco, CA, USA.
Methods in Molecular Biology (Clifton, N.J.)
|July 4, 2019
Summary
This study presents a rapid method using His-Glow fluorescent probes for optimizing integral membrane protein (MP) purification. This approach accelerates structural and functional studies of MPs, crucial for drug development.
Area of Science:
- Biochemistry
- Structural Biology
- Drug Discovery
Background:
- Integral membrane proteins (MPs) are vital drug targets but challenging to study due to difficulties in solubilization and purification.
- Current methods for optimizing MP production conditions are often time-consuming and labor-intensive.
Purpose of the Study:
- To develop a rapid and efficient strategy for identifying optimal solubilization and purification conditions for recombinant membrane proteins (MPs).
- To enable faster downstream structural and functional studies of MPs for drug development.
Main Methods:
- Utilized a noninvasive fluorescent probe (His-Glow) that binds to hexahistidine tags for fluorescent size-exclusion chromatography (FSEC).
- Employed a two-stage detergent screening strategy and a thermostability assay.
- Screened up to 96 condition/construct combinations in under 24 hours.
Main Results:
- Successfully enabled FSEC without green fluorescent protein (GFP) fusion, streamlining the process.
- The His-Glow method significantly accelerated the optimization of solubilization and purification conditions.
- Identified suitable constructs and conditions for large-scale MP production.
Conclusions:
- The His-Glow approach provides a streamlined and rapid strategy for optimizing recombinant MP production.
- This method facilitates parallel optimization for multiple MP targets, accelerating biochemical, immunization, and structural studies.
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