Reconstitution of a Reversible Membrane Switch via Prenylation by One-Pot Cell-Free Expression

Area of Science:

• Synthetic Biology
• Cellular Biophysics
• Protein Biochemistry

Background:

• Reversible protein membrane targeting regulates cellular networks like signaling and polarization.
• Reconstituting these "membrane switches" in vitro is crucial for designing minimal cells but remains challenging.

Purpose of the Study:

• To introduce a novel cell-free system for synthesizing and targeting prenylated proteins to membranes.
• To enable the in vitro reconstitution of key membrane-associated cellular functions for synthetic cell applications.

Main Methods:

• Developed cell-free prenylated protein synthesis (CFpPS) integrating synthesis and prenylation machinery.
• Engineered proteins with a C-terminal 4-peptide motif to confer membrane affinity.
• Utilized biomimetic membranes for robust production of prenylated proteins in proximity.

Main Results:

• CFpPS enables synthesis and membrane targeting of proteins in a single reaction.
• Successfully reconstituted the prenylated polarity hub Cdc42 and its regulator in vitro.
• Demonstrated robust production of soluble and membrane-associated prenylated proteins.

Conclusions:

• CFpPS is a versatile platform for engineering membrane-associated protein functions in synthetic cells.
• This method facilitates the implementation of cellular features like polarity induction in minimal cell designs.
• CFpPS overcomes previous limitations in reconstituting membrane switches in vitro.