Engineering protein thermostability using a generic activity-independent biophysical screen inside the cell

Area of Science:

• Biochemistry
• Biotechnology
• Structural Biology

Background:

• Protein stability is crucial for developing commercial proteins and biopharmaceuticals.
• Identifying mutations that enhance protein stability is challenging due to frequent neutral or deleterious outcomes.
• Current methods for assessing protein stability are often indirect and activity-dependent.

Purpose of the Study:

• To develop a high-throughput, direct, and biophysical method for screening intrinsic protein stability.
• To identify stabilizing mutations in diverse and challenging proteins, including biotechnologically relevant ones.
• To demonstrate the application of this method for improving the stability of protein drugs.

Main Methods:

• A high-throughput colony-based stability screen was developed.
• The screen provides a direct biophysical readout of intrinsic protein stability.
• The method was combined with random mutagenesis for identifying thermostable variants.

Main Results:

• Successful identification of thermostable variants across 10 diverse proteins.
• Included were a single-chain antibody, a commercial enzyme, and an FDA-approved protein drug.
• Thermostabilization of a protein drug led to significant improvements in its long-term stability.

Conclusions:

• The developed screening method is generic and activity-independent.
• This approach offers a powerful tool for enhancing protein stability in various applications.
• It facilitates the development of more robust biopharmaceuticals and proteins for research.