Supporting SPR Studies by Optimizing Protein Stability and Biotinylation

The Challenge

A client required a nuclear receptor protein to support surface plasmon resonance (SPR) studies using a Biacore platform.

Although the target had been produced previously and was commercially available, it was well known to present stability challenges. The client’s requirements extended beyond simple protein production. The final material needed to:

• Be suitable for SPR immobilisation.
• Contain a His-tag to support purification.
• Be site-specifically biotinylated for capture on the sensor surface.
• Maintain sufficient stability throughout the assay workflow.

The challenge was therefore to generate a high-quality, well-characterised protein preparation capable of supporting reliable SPR experiments.

Our Approach

Following a detailed review of the available literature and discussions with the client, a construct was designed that incorporated:

• Appropriate domain boundaries.
• A C-terminal 6His tag to support purification.
• An AviTag to enable site-specific biotinylation.

To generate fully biotinylated protein, the construct was co-expressed with biotin ligase, allowing intracellular biotinylation during protein production.

Given the well-documented stability issues associated with the target, the expression and purification strategy was developed around maintaining protein integrity. The protein was produced and purified in the presence of both:

• A stabilising ligand.
• A suitable detergent.

This approach was used to minimise aggregation and address the known stability challenges associated with the receptor.

Following affinity purification, a polishing size exclusion chromatography step generated milligram quantities of purified protein suitable for client studies.

The removal of detergent was also investigated to support potential use in alternative screening formats. While a second preparation could be generated, yields were significantly reduced as the underlying stability issues re-emerged.

Verifying Protein Quality

A comprehensive analytical characterization package was used to evaluate the final protein preparation.

Quality control demonstrated that the protein was:

• Greater than 95% pure by SDS-PAGE.
• 100% biotinylated as confirmed by intact mass spectrometry.

In addition, thermal shift analysis produced a clean and reproducible melting profile, providing further evidence that the protein preparation was suitable for downstream biophysical studies.

This combination of analytical approaches provided confidence that the material met the quality requirements for SPR applications.

Outcome

Using the optimized production and purification strategy, Sygnature Discovery successfully delivered stable, highly purified, site-specifically biotinylated protein for the client’s SPR programme.

The client subsequently reported a 20-fold increase in SPR assay window compared with material that had previously been used.

This improvement demonstrated how protein quality, stability, and biophysical suitability can directly influence downstream assay performance.

Why It Matters

Producing proteins for biophysical studies often requires more than achieving expression and purity targets. Protein stability, functional integrity, and compatibility with assay formats can all influence the quality of the resulting data.

This project demonstrates how a combination of construct design, stability optimisation, site-specific biotinylation, purification strategy, and analytical characterization can help generate proteins that are not only produced successfully but also perform effectively in downstream applications.

Key Results

✅ Nuclear receptor protein successfully produced for SPR studies

✅ Construct designed with 6His tag and AviTag

✅ Intracellular biotinylation achieved through co-expression of biotin ligase

✅ Greater than 95% purity achieved

✅ 100% biotinylation confirmed by intact mass spectrometry

✅ Protein stability supported through use of stabilizing ligand and detergent

✅ Clean, reproducible thermal shift profile obtained

✅ Client observed a 20-fold increase in SPR assay window