Long-Term Protein Synthesis with PURE in a Mesoscale Dialysis System.

Area of Science:

• Synthetic biology
• Biochemistry
• Biotechnology

Background:

• Cell-free systems, particularly the PURE system, face challenges with limited reaction duration and low protein yield.
• Existing custom dialysis methods for cell-free reactions often necessitate specialized equipment, hindering accessibility.
• Improving the lifespan and productivity of cell-free systems is crucial for their broader application.

Purpose of the Study:

• To develop and evaluate an easy-to-assemble, medium-scale dialysis system for cell-free reactions.
• To enhance protein yield and prolong the reaction lifespan of the PURE cell-free system.
• To demonstrate the system's stability and effectiveness using commercially available components.

Main Methods:

• Construction of a medium-scale dialysis system using readily available commercial components.
• Application of the dialysis system to the PURE cell-free system for protein synthesis.
• Periodic exchange of the feeding solution during the dialysis process.
• Quantification of Green Fluorescent Protein (GFP) yield and monitoring of reaction duration.

Main Results:

• Achieved a protein yield of 1.16 mg/mL of GFP using the PURE system with the developed dialysis method.
• Extended continuous protein synthesis for at least 12.5 consecutive days.
• Demonstrated excellent stability and prolonged reaction lifespan of the cell-free system.

Conclusions:

• The developed, easy-to-assemble dialysis system effectively overcomes the limitations of short reaction lifespans and low yields in cell-free systems.
• This approach offers a practical and accessible solution for enhancing cell-free protein production using standard components.
• The system's stability and high yield performance indicate significant potential for various synthetic biology applications.