A continuous cell-free translation system capable of producing polypeptides in high yield

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Cell-free protein synthesis (CFPS) systems offer a versatile platform for producing proteins.
• Traditional batch CFPS systems are limited by product inhibition and substrate depletion.
• Continuous flow systems aim to overcome these limitations for prolonged synthesis.

Purpose of the Study:

• To develop and characterize a continuous flow cell-free translation system.
• To assess the long-term stability and efficiency of both prokaryotic and eukaryotic CFPS.
• To evaluate the system's capacity for producing specific protein products.

Main Methods:

• Constructed a continuous flow cell-free translation system with feeding buffer and product removal.
• Tested both prokaryotic (Escherichia coli) and eukaryotic (wheat germ) cell-free systems.
• Used MS2 phage RNA, brome mosaic virus RNA 4, and synthetic calcitonin mRNA as templates.

Main Results:

• The continuous system maintained high, constant polypeptide synthesis rates for tens of hours.
• Prokaryotic and eukaryotic systems produced ~100 copies of viral coat proteins per RNA template over 20 hours.
• Both systems generated 150-300 copies of calcitonin polypeptide per mRNA template over 40 hours.

Conclusions:

• Continuous flow cell-free translation systems enable prolonged and efficient protein synthesis.
• These systems demonstrate robust performance in both prokaryotic and eukaryotic contexts.
• The developed system is suitable for sustained production of various polypeptides, including viral proteins and therapeutic peptides.