ATP Regeneration from Pyruvate in the PURE System

Area of Science:

• Biochemistry
• Synthetic Biology

Background:

• The Protein synthesis Using Recombinant Elements (PURE) system enables cell-free protein synthesis.
• Enhancing ATP regeneration is crucial for optimizing PURE system efficiency.

Purpose of the Study:

• To integrate a novel ATP regeneration system into the PURE system.
• To evaluate the impact of this new system on protein synthesis yield and reproducibility.

Main Methods:

• Incorporated pyruvate oxidase, acetate kinase, and catalase for ATP regeneration from pyruvate and phosphate.
• Tested the system's performance with varying phosphate concentrations.
• Assessed protein yield using mCherry as a model protein.
• Validated results across homemade and commercial PURE systems.

Main Results:

• Successfully generated acetyl phosphate for *in situ* ATP rephosphorylation.
• Achieved high protein yields (up to 233 μg/mL mCherry) with a combined ATP regeneration system, a 78% increase.
• Demonstrated that high phosphate concentrations (∼10 mM) do not inhibit protein synthesis.
• Showed reproducibility and compatibility with commercial PURExpress.

Conclusions:

• A novel, efficient ATP regeneration pathway was successfully integrated into the PURE system.
• This enhancement significantly boosts cell-free protein synthesis yields.
• The approach is rational, reproducible, and applicable to synthetic biology and synthetic cell construction.