High-throughput system for the thermostability analysis of proteins

Area of Science:

• Biochemistry
• Protein Engineering
• Biotechnology

Background:

• Protein thermal stability is crucial for function and application.
• Current methods for assessing protein stability are low-throughput and labor-intensive.
• Predictive models for protein stability are limited by data availability.

Purpose of the Study:

• To overcome throughput limitations in protein thermal stability analysis.
• To develop a rapid, plate-based system for protein expression, purification, and denaturation analysis.
• To enable high-throughput screening of protein variants for improved stability.

Main Methods:

• Leveraged high protein secretion efficacy of Brevibacillus bacteria.
• Implemented plate-scale purification methodologies for simultaneous sample processing.
• Integrated expression, purification, and thermal denaturation analysis on a per-plate basis.
• Analyzed 384 samples within a 4-day workflow.

Main Results:

• Successfully established a high-throughput system for observing protein denaturation.
• Analyzed 186 single mutants of a nivolumab single-chain variable fragment.
• Quantified melting temperatures (Tm) with variations from -9.3°C to +10.8°C compared to wild-type.
• Demonstrated practical application in protein stabilization and rational design.

Conclusions:

• The developed system significantly enhances throughput for protein thermal stability assessment.
• This method facilitates data-driven protein design and accelerates rational protein engineering approaches.
• Enables rapid identification of protein variants with enhanced thermal stability.