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Cell-free protein synthesis (CFPS) is a powerful way to explore the synthesis of proteins and other polymers outside of the cellular environment

By Fred Ward

Cell-free protein synthesis (CFPS) is a powerful way to explore the synthesis of proteins and other polymers outside of the cellular environment. By removing the often-constricting condition that cells must survive the experiment, a scientist is free to introduce more exotic and aggressive modifications to the translational mechanism. Currently, CFPS is implemented through two main techniques: cell extracts or recombinant systems. Extract-based systems, involve breaking open cells, removing the membrane debris, and then using the resulting slurry of cellular components to carry out a protein synthesis reaction. This is often easy and economical – it is can easily grow more cells to get more material for your reaction – but is limited in the control you have over the system. There are thousands of other small molecules, enzymes, and other factors in cells that can interfere with your reaction, and few of these are removed during the generation of extracts. For example, a scientist may be attempting to reassign a codon to incorporate an exotic new monomer into a biopolymer. Often, they are looking for only a tiny level of incorporation of this difficult monomer – you have to start somewhere! In a lysate based system, it is very difficult to remove the tRNAs, tRNA charging enzymes, and release factors that would conflict with the incorporation of the new monomer. Even a small amount of the unwanted, ‘contaminating’ reaction would overwhelm any signal from the desired but unfavorable new reaction. Natural translation mechanisms are very hard to out-compete!

To get around these problems, hard-working scientists have purified the 35+ necessary components for protein synthesis away from the rest of the cell, a preparation known as the PURE system. Now, researchers can carry out CFPS with only the components they desire, avoiding the cross-talk and unintended reactions that extract-based systems are plagued with. For the first time, is it straightforward for labs to study complex translation reactions with minimal necessary components, making it far easier to unravel complicated interactions and engineer new properties. However, the isolation of each of the 35+ components is itself is an involved process, making the total preparation outside the scope of most labs. Commercial mixes of the PURE system are available, but at substantial cost that limited their usefulness outside of small screens. This week, Barbora Lavickova and Sebastian J. Maerkl published a pre-print [1] outlining a greatly streamlined process for at-home PURE system preparation. Instead of purifying each component individually, they combine bacteria expressing tagged version each of the proteins into one master mix, lyse that mix to create a master solution of tagged PURE system proteins, and purify those many tagged proteins in one step in bulk. This technique condenses the 35+ purification processes into a single step, likely saving many person-months of work over the traditional process.

There have been other attempts to simplify PURE system production, but none have managed to bring the cost down without sacrificing the efficiency of the system. The ‘one-pot’ PURE system prep presented by Lavickova and Maerkl delivers nearly equivalent protein synthesis ability at less than 1/10th of the cost of the commercial PURE system. Finally, there may be a simple home PURE preparation, allowing labs everywhere to economically deploy recombinant in vitro translation in their research. One major question remaining to be answered is the modularity of this system: can tRNA charging enzymes and other factors be removed from the purification effectively? Studying highly inefficient unnatural substrate charging and translation is a powerful application of the PURE system, but trace amount of wild-type PURE protein contamination can render experiments incomprehensible. One concern is that the bulk purification method allows for too much ‘carry over’ of untagged translation proteins from the lysate. We are eager to prep our own one-pot PURE and see what it can do!

  1. Lavickova, B., and Maerkl, S.J. “A simple, robust, and low-cost method to produce the PURE cell-free system”. BioRxiv 420570 (2018) link