Holistic engineering of cell-free systems for the synthesis of supramolecular protein assemblies

Through an intercontinental collaboration, our team has demonstrated an enhanced cell-free protein system. The key innovation is a holistic synthetic and systems biology approach that engineers the proteome of the cell-free system.

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Our group has been working on re-engineering natural systems for un-natural functions for over a decade. One of our model systems is the protein nanocages. They are composed of discrete protein subunits that self-assemble into caged supramolecular structures of nanometer-size. Nature provides us with plenty of protein nanocages, which range from the iron storage protein, ferritin, the multi-enzyme complex, E2 of pyruvate dehydrogenase, the vault, to viruses. While they do not typically carry drugs or contrast agents for medical imaging, nor are they used for stabilizing emulsions, we have re-engineered them for these applications. Little did we know that it would also serve as a case study in our collaborators’ hands.
 
It was one of the evening gatherings following dinner at the GRC Biointerface Science. The corresponding author of the paper, Cheemeng Tan of UC Davis, presented what his cell-free systems could do, and I was impressed. At the time, we had been trying to produce nanocages that could escape the endosomes by displaying cell-penetrating peptides on its surface. Unfortunately, these constructs did not express well and the cell growth was stunted. We expected that they punctured the membrane of the expression host. The year was 2016, and it was the beginning of a long collaboration.
 
Following the Nature Chemical Biology paper published in 2018 (https://doi.org/10.1038/nchembio.2514), Cheemeng’s group has subsequently stumbled upon an idea of using a holistic approach for their cell-free system. Out of curiosity, they overexpressed the translation machinery that included initiation, elongation, and release factors in different pools of E. coli. They found that mixing the cell extracts resulted in synthetic circuits capable of better and efficient translation. Starting with an empirical approach, it took the group a good two years to figure out what caused the high performance of the cell-free system. They managed to reduce the important factors for the improvement of whole-cell extracts from 34 to 12, as described in this 2020 Nature Communications paper. They also found how the overexpression of these factors resulted in the proteome reprogramming of E. coli. Can the factors be further reduced to the minimum? Which specific master regulators control the global proteome reprogramming? They are working on getting to the bottom of it.
 
Armed with the holistic approach, they successfully produced deGFP at a quantity double that of a commercial system. The next question was to produce more complex structures, such as our supramolecular protein assemblies and the gene-editing nuclease Cas9. At this time, our original plan of expressing the cell-penetrating-peptide-tagged nanocages had been put on ice. Now with the enhanced cell-free system, we were back in business; this time with the untagged version. The challenge was managing a bi-continental collaboration, where samples needed to be shipped timely to avoid unnecessary transit delays. Maintaining the cold environment for the "cocktails" of whole-cell extracts was crucial in retaining their efficacy, as we realized. Our task was to check if proteins produced using this cocktail would still be functional. We managed to produce the multi-subunit protein, ferritin, which instantaneously self-assembled upon the addition of iron. The caged structure observed under transmission electron microscopy, in addition to the color change of the solution into brown, serve as proof that the proteins retain their functions when produced using this cocktail.
 
The success of the production and assembly of highly complex supramolecular protein structures, such as the protein nanocages, shows that the holistically-engineered cell-free system presents tremendous opportunities. An obvious one is high-throughput screening for prototyping protein synthesis. Synthesis of bigger and more complex proteins such as antibodies and transmembrane proteins are the natural next challenges for the system to address. Maybe one day, the capability can replace solid-state peptide synthesis and be used to produce synthetic large protein assemblies with tailored functionalities.

Sierin Lim

Associate Professor, Nanyang Technological University, Singapore

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