Week 9: Cell free systems

1.Explain the main advantages of cell-free protein synthesis over traditional in vivo methods, specifically in terms of flexibility and control over experimental variables. Name at least two cases where cell-free expression is more beneficial than cell production. The greatest advantage the cell-free protein synthesis has over in vivo methods is that the viability of the cell does not have to be maintained in order to maintain the viability of the protein synthesis. By allowing proteins to be grown in an open environment, greater control can be exercised over the key factors that produce the protein. Materials can be added to help protein production which may otherwise be toxic to cell populations. Scientists can control ion concentrations, cofactors and energy sources. Enhancing and prohibiting materials can also be added. Growing proteins in a cell-free environment also means protein production does not have to accommodate cloning of cells.

2.Describe the main components of a cell-free expression system and explain the role of each component. The main components of a cell-free expression system are:

The cell extract. The extract contains the key components required for transcription and translation and include:

• ribosomes for protein synthesis
• tRNAs (which helps ensure the right amino acid is inserted into the protein being synthesised) for amino acid delivery
• Aminoacyl-tRNA synthetases (charging tRNAs)
• Initiation, elongation, and termination factors.
• Sometimes endogenous RNA polymerases, which help synthesise complementary RNAs from existing RNA templates. Gent

DNA or mRNA template. These encode the proteins of interest. The DNA templates will require transcription through the use of RNA polymerase. An mRNA template does not need to be transcribed and only needs to be translated. Both templates are used to help guide the process of producing amino acids.

Energy source. This is needed to power protein synthesis and generally comes from ATP and GTP Amino acids. These provide the raw materials to help build proteins. Nucleotides. These are needed if protein synthesis begins with DNA rather than mRNA. Buffers and salts.

3.Why is energy provision regeneration critical in cell-free systems? Describe a method you could use to ensure continuous ATP supply in your cell-free experiment.

4.Compare prokaryotic versus eukaryotic cell-free expression systems. Choose a protein to produce in each system and explain why.

5.How would you design a cell-free experiment to optimize the expression of a membrane protein? Discuss the challenges and how you would address them in your setup.

6. Imagine you observe a low yield of your target protein in a cell-free system. Describe three possible reasons for this and suggest a troubleshooting strategy for each.