A Novel Method to Evaluate Ribosomal Performance in Cell-Free Protein Synthesis Systems

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Cell-free protein synthesis (CFPS) enables studying translation and protein folding with purified components.
• Quantifying active ribosomes and synthesis cycles is crucial for CFPS system performance characterization.

Purpose of the Study:

• To develop a direct and reliable method for measuring ribosomal activity in CFPS systems.
• To analyze the impact of ribosomal modifications on protein synthesis productivity.

Main Methods:

• Development of an enhanced-arrest peptide variant for ribosome stalling.
• Utilizing centrifugation techniques and fluorescence correlation spectroscopy (FCS) to assess ribosome activity.
• Employing two-colour coincidence detection and colocalisation microscopy for real-time analysis.

Main Results:

• The enhanced-arrest peptide variant achieved nearly complete ribosome stalling (~95%) in GFPem production.
• Independent effects of ribosomal modifications on activity and synthesis cycles were thoroughly investigated.
• Real-time monitoring of key productivity parameters at the single-ribosome level was demonstrated.

Conclusions:

• The developed method provides a highly reliable measure of ribosomal activity in CFPS.
• This technique allows for detailed characterization and optimization of CFPS systems.
• Single-ribosome level analysis offers unprecedented insights into protein synthesis dynamics.