Week 09 Lab: Cell Free

LAB QUESTIONS CELL FREE

I answered these in relation to lab document and FP where relevent.

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.

Cell-free systems allow direct control over reaction components without needing viable cells, making them ideal for expressing toxic proteins (like AMPs) and for rapidly iterating fusion protein designs like CBM27_RGD_MaSp1. Specifically chosen to bypass the living cells.

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

A cell-free system contains a cell extract with ribosomes and translation factors, a DNA template, amino acids, an energy regeneration system, and salts/cofactors, each providing the machinery, instructions, building blocks, fuel, and environment needed for protein synthesis.

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.

ATP is consumed faster than it is naturally recycled, so PURExpress uses phosphoenolpyruvate (PEP) and pyruvate kinase to continuously regenerate ATP from ADP and keep translation running.

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

E. coli-based PURExpress is used for CBM27_RGD_MaSp1 because it needs no glycosylation, while a mammalian cell-free system (e.g. HeLa extract) would be chosen for a protein like human erythropoietin that requires glycosylation to function.

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.

For membrane proteins, the main challenge is hydrophobic aggregation requiring lipid nanodiscs or detergents. Main challenge for MaSp1 however is repeat regions are prone to aggregation and beta-sheet stacking, would need to add chaperones like DnaK to the PURExpress reaction to assist correct folding. This will need to be part of my assay protocol.

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.

Poor template quality is fixed by re-purifying DNA and optimising concentration; ATP depletion is addressed by increasing PEP; and protein aggregation is reduced by adding protease inhibitors and chaperones.

References: HTGAA Spring 2026. Week 9 Lab: Cell-Free Systems. Available at: https://2026a.htgaa.org/2026a/course-pages/weeks/week-09/lab/index.html [Your final project documentation] CBM27_RGD_MaSp1 fusion protein construct design and PURExpress expression protocol, HTGAA Spring 2026.