Homework#9

PART A

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.

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Advantages

• Better control of system conditions:
• Precise regulation of variables such as DNA concentration, nucleotides, energy, and cofactors.
• Components can be easily introduced or relocated.
• Direct access to the environment:
  • In the absence of cell membranes, products, intermediates, or inhibitors can be more directly monitored and manipulated.
• Provides greater speed and scalability in production:
• CFPS reactions can be started quickly and efficiently optimized in parallel.
• Widely versatile, capable of adapting to a large number of samples. </aside>

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Cases

• Production of toxic proteins or proteins that would be difficult to express in conventional systems:
• Example: bacterial toxins or proteins with domains whose conformation hinders cell growth (e.g., cytotoxins).
• It has also been reported that these systems can aid in studies to identify new drug candidates for threats such as cancer, hepatitis, and malaria due to their high-speed expression platform. </aside>

Paper referred

Carlson, E. D., Gan, R., Hodgman, C. E., & Jewett, M. C. (2012). Cell-free protein synthesis: applications come of age. Biotechnology Advances, 30(5), 1185–1194. https://doi.org/10.1016/j.biotechadv.2011.09.016

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

   

• Components

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1. Crude Cell Extract: This comes from bacterial cells, wheat, and insects, among others. It also contains all the necessary machinery for vital biological processes such as transcription and translation (RNA polymerases, ribosomes, tRNA synthetase, etc.).

2. DNA or RNA template: This can be a plasmid or linear DNA, and its function is to contain the genetic information for transcription and translation.

3. 1. Energy regeneration system: This is the energy supply for the system, providing ATP and GTP for processes such as transcription, translation, and system maintenance

4. Free amino acids: These are the basic substrates for synthesizing proteins (NTPs: ATP, GTP, CTP, UTP) -> transcription.

5. Ions and salts (Mg2+, K): They provide stability to structures such as ribosomes and facilitate enzymatic activity.

6. Cofactors and buffers: They maintain pH and stabilize enzymes. </aside>

7. 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.

WHY?

Protein formation is a biological process requiring high energy demands and therefore requires multiple molecules of ATP and GTP during translation, transcription, and other cellular processes.

These systems, unlike living cells, cannot regenerate ATP on their own through metabolism. Therefore, an efficient and functional energy generation system is necessary.

Phosphoenolpyruvate (PEP)-based system.

• Mechanism: This system uses phosphate groups that act as donors, regenerating ATP from ADP through the use of the enzyme pyruvate kinase.
• Reaction: PEP+ADP→Pyruvate+ATP
• Limitations:
  • On a large scale, this system can be expensive.
  • Some compounds, such as pyruvate, can accumulate, which can alter the pH over the long term.
• Kim, D. M., & Swartz, J. R. (2001). Regeneration of adenosine triphosphate from glycolytic intermediates for cell-free protein synthesis. Biotechnology and Bioengineering, 74(4), 309–316. https://doi.org/10.1002/bit.1125
• Jewett, M. C., & Swartz, J. R. (2004). Mimicking the Escherichia coli cytoplasmic environment activates long-lived and efficient cell-free protein synthesis. Biotechnology and Bioengineering, 86(1), 19–26. https://doi.org/10.1002/bit.20026

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

   

   Examples

   • Prokaryotic CFPS
     • Firefly Luciferase: Simple enzyme, that does not require post-translational modifications. Easy to detect via luminescence
   • Eukaryotic CFPS:
     • **Factor VIII (Coagulation factor)**Therapeutic protein (Hemophilia A). Complex glycoprotein; needs correct folding and glycosylation

   Zemella et al., 2015. Chembiochem

   Carlson et al., 2012. Biotechnology Advances https://doi.org/10.1016/j.biotechadv.2011.09.016

   Stech, M., & Kubick, S. (2015). Cell-free synthesis meets antibody production. Biotechnology Journal, 10(5), 607–614. https://do.org/10.1002/biot.201400447

2. 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.

• Main challenges:

  • Their hydrophobic nature and the fact that they aggregate in the absence of a suitable lipid environment.
  • In addition to their need to insert into a bilayer for proper folding.

• Strategies

  Incorporation of membrane environments:

  1. Liposomes: These vesicles could be added to create structures similar to lipid bilayers.
  2. Mild detergents: These compounds would serve to provide solubility to hydrophobic domains without denaturation.

• Henrich, E., et al. (2015). Analyzing membrane protein–ligand interactions by cell-free expression and biolayer interferometry. Analytical Biochemistry, 477, 11–18. https://doi.org/10.1016/j.ab.2015.02.004

• Lyukmanova, E. N., et al. (2012). Cell-free production of integral membrane proteins for structural studies. Acta Naturae, 4(2), 35–43.