Week 9 HW: 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.

Main advantages (flexibility & control):

Open system: You can directly add/remove components (DNA, cofactors, salts, inhibitors). Precise control: You can tune Mg²⁺, ATP, amino acids, etc. Rapid expression: No need for cloning → transformation → growth. Toxic proteins: You can express proteins that would normally kill cells.

When CFPS is better than in vivo:

Producing toxic proteins (e.g., antimicrobial peptides) Studying protein variants quickly (high-throughput screening, mutant libraries) Incorporating non-natural amino acids Expressing membrane proteins without worrying about cell viability

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

Cell extract (lysate): Contains ribosomes, tRNAs, enzymes DNA or mRNA template: The blueprint for your protein Amino acids: Building blocks for protein synthesis Energy system (ATP, GTP + regeneration system): Fuels translation Salts (Mg²⁺, K⁺): Maintain ribosome stability and activity Cofactors (NAD⁺, CoA, etc.): Support metabolic reactions Enzymes (optional): Folding, disulfide bond formation, etc.

  1. 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.

Protein synthesis is an ATP-burning monster. If ATP runs out, your system basically gives up and stares into the void. Without regeneration translation stops quickly, and yield drops dramatically.

For instance, use of phosphoenolpyruvate (PEP) or creatine phosphate is valid as energy sources since these regenerate ATP via substrate-level phosphorylation

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

Prokaryotic systems (e.g., E. coli extract):

Fast, cheap, high yield Poor at post-translational modifications

Use case:

Produce enzymes like β-galactosidase → no complex folding/modifications needed

Eukaryotic systems (e.g., wheat germ, insect, mammalian extracts):

Slower, expensive Can do folding, disulfide bonds, glycosylation (depending on system)

Use case:

Produce antibodies or glycoproteins → need proper folding and modifications

  1. 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.

Challenges include aggregation, misfolding and insolubility. The strategies might include:

Add detergents Use liposomes or nanodiscs to mimic membranes Optimize Mg²⁺ and chaperones Lower temperature to improve folding

A design idea would be:

CFPS + nanodiscs + chaperones → allows co-translational insertion into a membrane-like environment

  1. 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 DNA template quality

Problem: degraded DNA or bad promoter Fix: use fresh plasmid, stronger promoter (e.g., T7), optimize codons

  • Energy depletion

Problem: ATP runs out Fix: improve regeneration system (PEP, glucose system)

  • Protein misfolding or degradation

Problem: aggregates or proteolysis Fix: add chaperones, reduce temperature, include protease inhibitors

  • Suboptimal ion concentrations

Problem: Mg²⁺/K⁺ imbalance kills ribosome activity Fix: optimize salt concentrations experimentally