Cell-Free Protein Synthesis

During the 1960’s Nirenberg and Matthaei deciphered the first codon (Codon: UUU, Amino Acid: Phenylalanine) and were able to show that RNA controlled the production of proteins. Nirenberg and Matthaei were able to extract the cellular machinery from E. coli responsible for protein production and demonstrated that proteins could be made when no intact living cell was present (1). Our previous blog The Smallest Factories in the World — E. coli discussed E. coli cells and how we use them in the lab. This blog will discuss how the components of those cells can be utilized to produce larger and more pure quantities of proteins in less time than traditional methods.

Micrograph depicting different colored fluorescent probes for the homeotic protein Ubx (green), the transmembrane protein sanpodo (red), and the transcription factor (blue). PC Dr. Andrea H. Brand https://wellcomecollection.org/works/k6q99za5

The Classic Batch Reaction Method

Protein production has been done in vivo since its induction in 1976. This requires the insertion of genetic material called a plasmid into a cell’s DNA. Plasmids are circular snippets of DNA that cells naturally take up and institute into their genetic code for more diversity. Scientists have hijacked this phenomenon by creating synthetic plasmids — called vectors — which are inserted into cells via heat shock or electroporation. Cells that take up the vector are separated and grown into large colonies. When the colonies reach a sufficient size, they are induced to produce the target protein in abundance by activating the inserted gene. These colonies are lysed (torn open) and the cellular extract is gathered. The extract contains a variety of contaminants including many proteins aside from the target, so it must be purified — a process so involved, we wrote an entire blog about it: Protein Purification. The Classic Method requires a large initial time commitment to work out a viable protocol for each step. Even after the protocol is in place, the operation may be hampered by difficult uptake, slow cell growth, toxic proteins, poor yields, etc.

Proteins Produced Without Living Cells

Currently, a Cell-Free Protein Synthesis (CFPS) reaction consists of DNA, an energy source (energy-providing molecules ATP & GTP), ample amounts of amino acids, other cofactors such as magnesium, and cellular extract. This cellular extract is obtained the same way as the Classic Method — cells are lysed and debris is removed through centrifugation. However, the leftover components such as the molecular energy sources and amino acids which are usually removed in the Classic Method, can be utilized and supplemented — allowing for longer reactions times and fewer contaminant proteins. Classic Batch reactions are limited by depleting energy sources and bioavailable molecules (amino acids and ribonucleotides). This ultimately forces the reaction into limited protein yields. Cell-Free reactions are easier to scale and handle than those done in living cells. Continuous-flow cell-free (CFCF)(4) and continuous exchange cell-free (CECF) (5,6) provide longer reaction lifetimes and much higher protein yields. The theoretical maximum protein yield from the Classic Method is between 150 micrograms and 3 milligrams per liter (7), depending on the percentage of total protein in cell extract. The theoretical maximum protein yield for Cell Free Methods is several grams per liter(4)!

Comparison of cell-free and in vivo protein synthesis methods. (2)

The appreciation for CFPS reactions arise from the lack of a functional genome and therefore are not restricted to the cell’s life objective — meaning cells restrict production to preserve their normal functions and conserve energy. Additionally, proteins that may be toxic to cellular life cannot be produced in large amounts within cells. By removing the cell, these inhibitions are gone. High throughput could become much faster because the time required for cloning, growing, and purifying cells is non-existent. Proteins can be rapidly produced in satisfactory yields and purities. Not only will this allow for the faster accumulation of data and learning, but also reducing the amount of wasted resources for bacterial growth.

Another perk of CFPS is the ability to include non-natural amino acids in proteins. This will lead to new biochemical properties that can be explored and tested. Essentially, this means proteins can be synthesized that exhibit longer shelf life, stronger binding, as well as other unique characteristics not yet explored (2,3).

The Cell-Free Future

Cells have been used as the standard for protein production since the introduction of the process in the 70s. There have been many improvements to cell lines, reagents, and equipment, but none of these improvements have been able to make impactful changes to remove the bottleneck of growing cells. At the same time, there have been huge strides in computational power creating the necessity for big data — a necessity the Classic Method has not been able to meet. CFPS shows great potential and promises to fill the gap between analysis and production. The next decade may finally see E. coli disappear as the standard for protein production.

Links and Citations:

1. The Dependence of Cell-Free Protein Synthesis in E. coli Upon Naturally Occurring or Synthetic Polyribonucleotides. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC223178/?page=1
2. A User’s Guide to Cell-Free Protein Synthesis https://www.mdpi.com/2409-9279/2/1/24
3. Prolonging Cell-Free Protein Synthesis with a Novel ATP Regeneration System https://www.ncbi.nlm.nih.gov/pubmed/10577472/
4. A Continuous Cell-Free Translation System Capable of Producing Polypeptides in High Yield https://www.ncbi.nlm.nih.gov/pubmed/3055301/
5. A Semicontinuous Prokaryotic Coupled Transcription/Translation System Using a Dialysis Membrane https://www.ncbi.nlm.nih.gov/pubmed/8879155/
6. New England Biolabs, Inc. https://www.neb.com/tools-and-resources/usage-guidelines/protein-data

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