Community-Wide Experimental Evaluation of the PROSS Stability-Design Method

Area of Science:

• Protein Engineering
• Computational Biology
• Biotechnology

Background:

• Protein design methods have advanced, but often require expert knowledge.
• The PROSS algorithm aims to improve protein stability and expression levels.
• Widespread adoption of protein design tools is limited by complexity.

Purpose of the Study:

• To benchmark the PROSS algorithm as a stand-alone tool for protein scientists with limited modeling experience.
• To assess the general applicability of PROSS across diverse protein targets.
• To evaluate PROSS designs for expression, stability, and activity.

Main Methods:

• PROSS algorithm applied to 14 unrelated protein targets by 12 laboratories.
• Designs evaluated for heterologous expression in prokaryotic and eukaryotic systems.
• Thermal stability and activity assays performed on selected targets.

Main Results:

• Increased heterologous expression observed in 9 out of 14 targets.
• Improved thermal stability noted in 9 out of 10 tested targets.
• PROSS designs enabled expression of previously unexpressible proteins (hSCF, CLD12).

Conclusions:

• PROSS can enhance stability and expressibility in diverse proteins.
• Target-specific expression conditions are typically required for optimal PROSS performance.
• Community-wide benchmarking demonstrates PROSS's utility and identifies areas for future algorithm development.