In 2020, 6.9 million tonnes of plastic ended up in landfills. A circular economy offers a framework for integrating sustainability into the plastic industry. Protein-based materials are an emerging field with the potential to replace plastics thanks to their modular design and multiple applications. Proteins are sequence-defined polymers, consequently, different sequences from the same building blocks can yield various functionalities. In nature, protein metabolism offers a unique example of circular recycling. Protein mixtures are hydrolysed into their building blocks, the amino acids (AAs). These AAs can be reinserted into a new synthesising protein, thus being circularised from an initial protein mix to a new protein tailored to the organism's needs. Protein metabolism shows a circular recycling scheme between different functional protein materials, something that is currently a challenge for other types of functional polymeric materials.

These processes were reproduced in vitro in our previously published work called NaCRe (Nature-inspired Circular Recycling). It was a proof-of-concept for a novel approach to the circular recycling of soft materials, relying on an emerging class of polymeric materials which are proteins. A mixture of three proteins was incubated in a two-step in vitro enzymatic digestion. This produced a pool of AAs that was then introduced into a reconstituted in vitro protein synthesis system lacking AAs to produce a different protein. This showed that different proteins could follow a circular recycling round in vitro in a diluted solution and in the microgram scale. Functionalities were achieved in the recycled protein that were not present in any of the proteins from the initial mix, and circularity was proven by a second round of recycling which provided another perfectly functional protein.

The first part of the thesis shows that NaCRe can be scaled up from the microgram scale to the milligram scale, expanding it beyond diluted solutions to macromolecular assemblies. Proteins are digested into amino acids, and fed into a cell-free expression system to produce recycled muGFP. The protein is then cross-linked into the recycled hydrogel (~1 mg) with a 20% efficiency, demonstrating that NaCRe can be run at a milligram scale and that the functionality of the recycled material can differ from the initial material. Additionally, an elastomeric resilin-like protein is expressed using commercial cell-free E. Coli lysate. This suggests that this elastomeric protein could be produced through NaCRe.

NaCRe relies on cell-free expression, an expensive method with low yields. One strategy to address these limitations is to extend the reaction lifetime. In the second part of the thesis, the cell-free PURE system (Protein synthesis Using Recombinant Elements) is employed to investigate the lifetime of E. Coli's transcription and translation machinery when by-products are removed and building blocks and energy molecules are supplied. We used a custom-designed plate system supporting 100 uL reactions in a 1 mL dialysis chamber. The plate allows direct access to both chambers for sampling, imaging, and fits in standard plate-readers. This provides in-time fluorescence monitoring to reveal the kinetics of the expression reaction. Through periodic exchange of the dialysis chamber's contents, we extend the reaction time from 4 to 12.5 days, significantly increasing protein yields and exploiting PURE's stability.