Efficient and scalable method for scaling up cell free protein synthesis in batch mode

Area of Science:

• Biotechnology
• Molecular Biology
• Protein Expression

Background:

• Cell-free protein synthesis (CFPS) offers advantages for rapid protein production.
• Scaling up CFPS reactions in batch mode presents challenges in maintaining volumetric yields.
• Efficient scale-up methods are crucial for the industrial application of CFPS.

Purpose of the Study:

• To introduce and evaluate a novel thin film method for scaling up batch mode cell-free systems.
• To assess the performance of the scale-up method using two different E. coli-based cell-free systems.
• To determine the impact of the method on the expression of chloramphenicol acetyl transferase (CAT) and a GMCSF-scFv fusion protein (GLH).

Main Methods:

• Applied a novel thin film batch mode scale-up method to E. coli-based cell-free systems.
• Tested reaction volumes ranging from 15 to 500 microL for CAT and GLH protein expression.
• Compared protein yields obtained with the thin film method against traditional test tube reactions.

Main Results:

• The thin film scale-up method consistently preserved total, soluble, and active volumetric yields for both CAT and GLH.
• At 500 microL, the PANOx SP system achieved significantly higher active CAT (560 microg/mL) and GLH (99 microg/mL) yields compared to test tubes (250 microg/mL and 72 microg/mL).
• The Cytomim system also showed substantial improvements with the thin film method, yielding 481 microg/mL active CAT and 109 microg/mL active GLH versus 29 microg/mL and 5 microg/mL in test tubes.

Conclusions:

• The novel thin film approach is a generalizable scale-up technology for cell-free protein synthesis.
• Improved oxygen supply and increased hydrophobic surface availability contribute to enhanced protein expression and folding.
• This method is suitable for various protein targets, cell-free systems, and reaction volumes, facilitating broader application of CFPS.