Membrane proteins underlie numerous human pathologies and are critical components of various drug-delivery vehicles, including nanoparticles and exosomes. However, the study and use of these proteins remain limited because of our inability to synthesize them. Current methods rely on brute force synthesis using living cells, which often fail despite relentless optimization. To circumvent the shortcomings of cell-based synthesis, we sought to create a novel high-throughput platform to rapidly synthesize and insert membrane proteins into liposomes using cell-free protein synthesis.We first set out to develop a novel cell-free protein synthesis system that would enhance our ability to produce proteins. Our objective was to bias the resource allocation of the host cells used in the whole-cell extract preparation to prioritize the synthesis of proteins. We took a holistic approach to our engineering efforts, wherein we exploited the natural crosstalk between the metabolic network in the cells and the artificial genetic modules that we designed. Specifically, we show that local modules expressing translation machinery can reprogram the bacterial proteome, changing the expression levels of more than 700 proteins. The resultant feedback generates a cell-free system that can synthesize fluorescent reporters, protein nanocages, and the gene-editing nuclease Cas9, with up to 5-fold higher expression than classical cell-free systems. Our work demonstrates a holistic approach that integrates synthetic and systems biology concepts to achieve outcomes not possible by only local, orthogonal circuits.
Next, we shifted to developing a system to test a wide array of different cell-free reactions. This task requires rapid, ultra-low volume handling operations. To address this, we developed a pipette-free robotic-microfluidic interface using a microfluidic-embedded printing cartridge to achieve seamless integration of nanoliter scale liquid handling and robotic automation. Our microfluidic adaptive printing system incorporates on-deck calibrations and real-time monitoring to achieve nanoliter precision in droplet volumes and reproducible droplet generation across thousands of droplets. The optimized system can assemble unique cell-free reactions across a 384-well plate utilizing more than ten different reagents—equivalent to approximately 7,500 droplets—in under an hour.
Finally, we combine our cell-free protein synthesis system and our droplet printing robot to tackle membrane protein synthesis. Specifically, we first create a high-throughput workflow, combining a novel fluorescent reporter, cell-free protein synthesis, and nanoliter droplet printing, to enable 384-well plate-based synthesis of membrane proteins. Based on the high-throughput platform, we generate the first big data on membrane protein synthesis from over 20,000 different synthesis reactions, covering 28 membrane proteins, and varying lipid types, chemical environments, and chaperone proteins. Using this wealth of data, we implement an active-learning algorithm to synthesize membrane proteins that have not been synthesized in the literature. We also identify structural descriptors of each protein that allow for accurate prediction of a protein’s likelihood to be successfully synthesized. Our new platform and dataset of membrane-protein synthesis overcome a major barrier in the field of membrane proteins, enabling rapid study and design of membrane proteins in future work.
