Evaluation of Membrane Protein Properties by Fluorescence-Detection Size-Exclusion Chromatography (FSEC) Using an HPLC System

Significance of the Topic

Cell membranes host a variety of proteins essential for signal transduction, substance transport, and serve as prime drug targets. However, structural studies are hindered by the challenge of producing sufficient quantities of stable membrane proteins. Fluorescence-detection size-exclusion chromatography (FSEC) on an HPLC platform enables rapid assessment of expression, stability, and homogeneity of membrane proteins in unpurified extracts, addressing key bottlenecks in structural biology.

Objectives and Study Overview

This application note describes the implementation of FSEC using a Shimadzu HPLC system at the Nureki Laboratory, University of Tokyo. The goals are to present the system configuration, outline the sample preparation workflow, and showcase examples of FSEC applications for selecting membrane protein constructs suitable for structural analysis.

Methodology and Instrumentation

FSEC Principle:

• GFP is fused to the N- or C-terminus of target membrane proteins expressed in HEK293 or Sf9 cells.
• Cells are solubilized in detergent-containing buffer and analyzed without prior purification by gel filtration chromatography.
• Detection of GFP fluorescence (Ex 480 nm, Em 510 nm) isolates the target signal from background proteins.

Sample Preparation Workflow:

1. Harvest cells by low-speed centrifugation.
2. Resuspend pellet in buffer (e.g., 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.03% DDM) and mix at 4 °C for 30–60 min.
3. Ultracentrifuge to remove insoluble debris; transfer supernatant to autosampler vials.
4. Inject 30–100 µL into a size-exclusion column; elute at 4 °C with controlled flow.

Used Instrumentation:

• Shimadzu Prominence HPLC with CBM-20A controller.
• LC-20AD solvent delivery pump (four lines A–D).
• SIL-20AC autosampler for 70 or 105 vials.
• FCV-14AHi six-port column switching valve.
• RF-20Axs fluorescence detector (Ex/Em 480/510 nm for GFP; 280/350 nm for tryptophan).
• Columns: Bio-Rad ENrich SEC 650, GE Superdex 200 Increase 5/150, Superose 6 Increase 10/300.

Key Results and Discussion

• Species Screening: Four eukaryotic transporters displayed distinct GFP-FSEC profiles; one construct produced a sharp peak indicating high expression and monodispersity.
• Thermostability (FSEC-TS): Heat treatment followed by FSEC reveals denaturation temperatures through peak height reduction and aggregation.
• Tryptophan-FSEC (Trp-FSEC): Monitoring intrinsic Trp fluorescence at ng levels enables characterization of purified samples and receptor–antibody complexes.
• NTA-Probe FSEC: Fluorescent NTA peptides bind His-tagged targets, providing an alternative to GFP for detecting proteins in unpurified samples.

Benefits and Practical Applications

• Rapid screening of expression level, stability, and aggregation state directly in crude lysates.
• High sensitivity fluorescence detection supports low-abundance targets.
• Automated sample handling and column switching increase throughput and experimental reproducibility.
• Adaptable to constructs unsuitable for GFP tagging and to various protein–ligand or complex formation studies.

Future Trends and Opportunities

• Integration with cryo-EM and other structural workflows to accelerate candidate selection.
• Development of novel fluorescent probes for expanded target detection.
• High-throughput FSEC platforms for automated optimization of buffer, detergent, and lipid conditions.
• Extending applications to membrane protein complexes and transient interaction screening.

Conclusion

FSEC on a customized HPLC system offers a powerful, efficient approach to characterize membrane protein behavior early in structural biology pipelines. By enabling high-throughput, sensitive analysis of unpurified samples, this platform streamlines the identification of stable, homogeneous constructs, thereby accelerating downstream crystallography and cryo-EM studies.

References

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