CFPS Systems

General Questions

Reference paper: Cell-Free Gene Expression: Methods and Applications

1. Main advantages of cell-free protein synthesis (CFPS) systems over traditional in vivo methods

According to the course material, CFPS systems offers more flexibility because there is no need anymore to maintain cells alive. For instance, it allows the production of toxic proteins that would otherwise kill the cells producing them. Using easier cell machinery systems can also give scientists more flexibility in adapting their experimental design. For instance, it allows them to incorporate non-natural amino acids into the reaction. And finally, CFPS systems also offer much easier storage conditions because it is possible to freeze-dry the reactions on tiny pellets.

Cell-free protein synthesis also offers more control during production: there is no unknown cellular process occurring in parallel, nor interference with other proteins naturally produced by the cells. The conditions of the environment (e.g. ions concentrations, pH, enzymes etc.) can be defined more precisely. Finally, it makes it much easier to isolate the target protein. These assets are useful in the pharmaceutical industry for producing large batches of hormones (e.g. insulin), vaccines or medicines.

To go further…

A critical comparison of cellular and cell-free bioproduction systems

Cell-Free Synthesis: Expediting Biomanufacturing of Chemical and Biological Molecules

2. Main components of a CFPS system

Components of a CFPS reaction mixed in a test tube:

From Cell-free protein synthesis and vesicle systems for programmable therapeutic manufacturing and delivery

The CFPS system mixture contains:

• DNA template (linear or plasmid): gene of the protein of interest
• Cell extract that contains RNA polymerase, enzyme that synthesizes RNA from DNA; ribosomes, molecular machines that reads the mRNA and assembles the amino acids into a protein, transfer RNAs, adapter molecules that carry amino acids and match them to the corresponding codons on the mRNA during translation and further enzymes needed for the protein synthesis.
• Amino acids: building blocks of the proteins
• Free nucleotides: building blocks of the mRNA (A,C,G, U)
• Co-factors: maintain enzyme activity and ensure proper protein folding.
• Energy source: ATP, GTP and their regenerating substrates (e.g. PEP) provide the energy required for both transcription and translation processes.
• Salts: provide the ions needed for optimal enzyme activity and structural stability of the system.

3. Energy regeneration in cell-free systems

The translation and transcription processes require a lot of energy. Without a continuous supply in ATP, the protein synthesis quickly shuts down. Thus, energy regeneration is needed to provide a continuous supply of energy and avoid the accumulation of by-products (e.g. phosphate) that can interfere with the protein synthesis.

There are many different ways to regenerate energy in cell-free systems: see references. Choosing glucose as an energy source can be interesting in the prototyping phase of a project because it is a highly cost-effective and an efficient way to ensure a continuous supply of ATP via glycolysis in both E. coli and yeast extracts. It can also be combine with creatine phosphate in a dual system: creatine phosphate acts as a high-energy phosphate donor for rapid ATP regeneration via creatine kinase, while glucose feeds the glycolytic pathway to produce ATP and consume inorganic phosphate.

From The cost-efficiency realization in the Escherichia coli-based cell-free protein synthesis systems

References

ATP Regeneration from Pyruvate in the PURE System

Cell-Free PURE System: Evolution and Achievements

Development of prokaryotic cell-free systems for synthetic biology (includes PANOx energy regeneration system)

4. Prokaryotic vs. eukaryotic cell-free expression systems

Prokaryotic cell-free systems are fast and efficient for producing simple proteins. For instance, E.coli systems are ideal for producing simple proteins such as Green Fluorescence Protein (GFP) in labs and in the industry.

On the other hand, eukaryotic systems are slower and more expensive, but they can be used for the production of proteins which require complex folding, disulfides bridges, and post-translational modifications (e.g. glycosylation, lipidation) such human anti-bodies.

Reference

Protein Synthesis in Prokaryotes vs. Eukaryotes: What’s the Difference?

Cell-Free Protein Synthesis: Pros and Cons of Prokaryotic and Eukaryotic Systems

5. Cell-free systems for the synthesis of membrane proteins

Membrane proteins are composed of transmembrane domains which makes them hydrophobic and thus hard to express in traditional cell-based systems: they need a special environment to avoid aggregation, and enable the proteins to fold and function correctly. However, it is possible to recreate such membrane-like environments by adding specific supplements to the reaction depending on the protein (Is it small and simple, or bigger and complex?) and what we want to do with it (do we only want to extract the protein or does it have to be functional?).

Cell-free systems derived from prokaryotic, as well as eukaryotic extracts that lack endogenous microsomes (natural membrane fragments from the cells) be can be supplemented with:

• Detergents, commonly used for solubilization and fast extraction membrane proteins.
• Nanodiscs, small membrane-like structures of adaptable size and easy to purify. Useful for stabilizing and studying the structure of the proteins.
• Liposomes, tiny vesicles composed of bilayer membranes that are more difficult to purify but also more adapted for transporter proteins and the ones that need to be oriented in the membrane. Useful for testing the function of the proteins.

From Cell-free Membrane Protein Synthesis

For my final project “Sensing perimenopause: a bioluminescent art installation”, one design strategy is to use the G protein–coupled receptor to elicit a bioluminescent response to an environmental change, e.g. a change in extracellular levels of glycogen in the vaginal secretions. If using a cell-free system, I might want to use eukaryotic cell extracts and add liposomes to ensure that the receptor can sense the glycogen levels in real-time.

To go further…

Cell-Free Protein Synthesis: A Promising Option for Future Drug Development

Membrane protein synthesis in cell-free systems: From bio-mimetic systems to bio-membranes

Membrane protein production in Escherichia coli cell-free lysates

Membrane protein synthesis in cell-free systems: From bio-mimetic systems to bio-membranes

Cell-free synthesis of membrane proteins: Tailored cell models out of microsomes

6. Troubleshooting Low Protein Yield

Achieving optimal protein yield is a major challenge in cell-free protein synthesis systems. Below is a table that lists three common issues that can lead to low protein production and how to solve them.

References

How to Troubleshoot Low Protein Yield After Elution

Solved: Low Yields in Cell-Free Protein Synthesis

Troubleshooting Protein Folding Issues in Cell-Free Synthesis: Tips from Industry Experts