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 protein synthesis (CFPS) systems were created as a way of reducing the complexities of in vivo methods, reducing time, elements and making the system more controllable. One of the biggest issues that was tackled was the presence of a cell membrane, allowing for protein synthesis elements to be easily accessible and not having to go through the normal transport systems to get to them. The barrier also made experiments harder to standardise, created compatibility issues, and added unwanted variability (Khambati, et al., 2019).
Cell-free systems are overall considered better for protein production due to their high controllability, tolerance, stability, and the fast protein production.
Two ideal cases for the use of CFPS would be the production of toxins, which are usually hindered by the effect they have on the producing cell, or unstable/proteolytically sensitive proteins. But more interesting, they are vey useful for the incorporation of unnatural/non-canonical amino acids or work that include the expansion of the genetic code.
Describe the main components of a cell-free expression system and explain the role of each component.
The CFS I am going to use as an example is the PURE system (Protein synthesis Using Recombinant Elements), first designed by Shimizu, et al., in 2001. the system, based on the E. coli translation apparatus, is devoid of proteases, nucleases, and membrane proteins, which usually are present in a cell extract and hinder the proper measurement of synthesised proteins.
The system is composed by a mixture of all necessary translation elements, including Initiation factors, elongation factors and release factors, corresponding to the 3 steps of protein synthesis; as well as all aminoacyl-tRNA synthetases, in charge of correlating a codon to its corresponding amino acid. It also includes methionyl-transformilase for the initial methionine at the start of protein synthesis, T7 RNA polymerase and highly purified ribosomes. And finally, all the required substrates, such as tRNAs, NTPs, all the amino acids, and enzymes catalysing phosphate metabolism.
| PURE system components A | Conc. | PURE system components B | Conc. |
|---|---|---|---|
| Buffer mix (×2) | |||
| L-Alanine | 0.6 mM | L-Threonine | 0.6 mM |
| L-Arginine | 0.6 mM | L-Tryptophan | 0.6 mM |
| L-Asparagine | 0.6 mM | L-Tyrosine | 0.6 mM |
| L-Aspartic acid | 0.6 mM | L-Valine | 0.6 mM |
| L-Cysteine | 0.6 mM | HEPES-KOH (pH 7.6) | 100 mM |
| L-Glutamine | 0.6 mM | Potassium glutamate | 200 mM |
| L-Glutamic acid | 0.6 mM | Mg(OAc)2 | 26 mM |
| L-Glycine | 0.6 mM | Spermidine | 4 mM |
| L-Histidine | 0.6 mM | DTT | 2 mM |
| L-Isoleucine | 0.6 mM | Creatine phosphate | 40 mM |
| L-Leucine | 0.6 mM | ATP | 6 mM |
| L-Lysine | 0.6 mM | GTP | 6 mM |
| L-Methionine | 0.6 mM | CTP | 2 mM |
| L-Phenylalanine | 0.6 mM | UTP | 2 mM |
| L-Proline | 0.6 mM | FDa | 20 µg/ml |
| L-Serine | 0.6 mM | tRNA mix | 112 OD260/ml |
| Enzyme mix (×10) | |||
| Ala RS | 688 μg/ml | Tyr RS | 6 μg/ml |
| Arg RS | 20 μg/ml | Val RS | 8 μg/ml |
| Asn RS | 220 μg/ml | MTF | 200 μg/ml |
| Asp RS | 80 μg/ml | IF1 | 100 μg/ml |
| Cys RS | 12 μg/ml | IF2 | 400 μg/ml |
| Gln RS | 38 μg/ml | IF3 | 100 μg/ml |
| Glu RS | 126 μg/ml | EF-G | 500 μg/ml |
| Gly RS | 96 μg/ml | EF-Tu | 1 mg/ml |
| His RS | 8 μg/ml | EF-Ts | 500 μg/ml |
| Ile RS | 395 μg/ml | RF1 | 100 μg/ml |
| Leu RS | 40 μg/ml | RF2 | 100 μg/ml |
| Lys RS | 64 μg/ml | RF3 | 100 μg/ml |
| Met RS | 21 μg/ml | RRF | 100 μg/ml |
| Phe RS | 165 μg/ml | MK | 30 μg/ml |
| ProRS | 102 μg/ml | CK | 40 μg/ml |
| Ser RS | 19 μg/ml | NDK | 11 μg/ml |
| Thr RS | 63 μg/ml | PPiase | 10 μg/ml |
| Trp RS | 11 μg/ml | T7 RNA polymerase | 100 μg/ml |
| Ribosome (×20) | |||
| 70S ribosome | 20 μM |
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10-Formyl-5,6,7,8-tetrahydrofolic acid. RS, tRNA synthetase; MTF, methyltetrahydrofolate; IF1, Initiation factor 1; EF-G, Elongation factor G; EF-Tu, Elongation factor Tu; EF-Ts, Elongation factor Ts; RF1, Release factor 1; RRF, Ribosome recycling factor; MK, myokinase; CK, creatine kinase; NDK, nucleoside-diphosphate kinase; PPiase, peptidylprolyl-isomerase.
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Table taken from https://www.nature.com/articles/nprot.2015.082/tables/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.
Ensuring a proper energy pool in cell-free systems is essential for the process to work, as the system requires ATP to work. A balanced phosphate cycle is needed for the protein synthesis to last longer and be more efficient. This has proven to be an tricky process, and has been a research topic for decades (https://pubmed.ncbi.nlm.nih.gov/10577472/, https://pubmed.ncbi.nlm.nih.gov/17634594/, https://pmc.ncbi.nlm.nih.gov/articles/PMC4651010/)
A possible way to ensure continuous ATP supply would be via automated introduction of it in the system at fixed intervals, or maybe developing an optimised set of enzymes that would be highly efficient at catalysing phosphate groups, ensuring the proper circulation of ATP when needed. Another alternative would be a continuous flow system, ensuring the substrate and elements remain flowing while the protein of interest is retained.
Compare prokaryotic versus eukaryotic cell-free expression systems. Choose a protein to produce in each system and explain why.
Classic CFS include E. coli, wheat, insect cells, **rabbit, and human. Each system has advantages, such as E. coli being the cheapest and with the highest yield, but unable to perform post-translational modifications. Wheat is the highest producer out of the eukaryotic CFS, but is lacking with certain protein modifications. Each system should be used on a case-by-case basis.
