Performance benchmarking of four cell-free protein expression systems

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Expression

Background:

• Cell-free protein expression systems are crucial tools in various biological applications.
• Despite known performance differences, systematic comparisons of these systems are scarce.

Purpose of the Study:

• To systematically evaluate and compare the performance of four distinct cell-free protein expression systems.
• To assess expression yield, protein integrity, and aggregation propensity across different systems.

Main Methods:

• Expressed 87 N-terminally GFP-tagged human cytosolic proteins across E. coli, wheat germ (WGE), HeLa, and Leishmania-based (LTE) cell-free systems.
• Utilized single-molecule fluorescence spectroscopy, SDS-PAGE, and Western blot analysis for characterization.
• Quantified expression yields, full-length product fraction, and aggregation levels.

Main Results:

• E. coli exhibited the highest expression yields but produced significant truncated proteins (>70 kDa) and high aggregation.
• Wheat germ extract (WGE) was the most productive eukaryotic system.
• HeLa and Leishmania-based extract (LTE) systems yielded lower protein amounts but demonstrated superior protein integrity and lower aggregation.

Conclusions:

• The choice of cell-free system significantly impacts protein expression yield, integrity, and aggregation.
• E. coli is suitable for high-yield production but may require optimization for protein quality.
• HeLa and LTE systems are advantageous for producing high-quality, aggregation-resistant eukaryotic proteins suitable for direct analysis.