Engineering Agar Hydrogel Beads to Control Encapsulation and Release of Model Proteins for Cell-Free Proteins Synthesis

Synthetic cells often refer to self-assembled soft matters in cell-like vesicle structures or multi-compartmentalized organelles, which are designed for mimicking the behavior, function, and structure of a living cell. Protein synthesis is one of the essential processes in living cells, which involves transcription and translation. Therefore, enabling in vitro transcription and translation (IVTT) for cell-free protein synthesis (CFPS) in synthetic materials would be a step-stone toward the development of synthetic cells. Hydrogel platforms have been highlighted as promising biomaterials for therapeutic applications due to their stability, binding affinity, release rates, and target ability. Agar is reported to be the most efficient hydrogel for CFPS due to its high strength at low concentration, low viscosity, high transparency, and sharp setting temperature. We aim to develop a synthetic cell model by using agar hydrogel beads (AHBs) that allow CFPS and controlled release of the protein building blocks synthesized via IVTT. In this presentation, I will share the synthesis methods of AHBs that we have optimized to obtain different sizes from 200 nm to 2µm. We controlled the average AHB size by tuning the homogenizer speed and time during emulsification. With AHBs with an average diameter of 1 ~ 2 µm, I studied the mechanism of encapsulation and release of model fluorescent proteins (enhanced green fluorescent protein (eGFP-Z_E) and red fluorescent protein (mCherry-Z_E)). I hypothesized that the fluorescence intensity profile released from AHBs in the solution would be affected by the initial concentration of the proteins and encapsulation yield of AHBs. Next, I investigated the changes in the release profile and rate of the model proteins from AHBs upon the introduction of agarase, which is an enzyme that catalyzes the hydrolysis of agar. Based on the results, I will design the experiments to imbibe CFPS agents of eGFP into AHBs to study CFPS yield in AHBs and trigger the release of de novo protein building blocks made in this system. Finally, this work will be expanded to enable CFPS of recombinant fusion protein building blocks that can self-assemble into either membrane-less organelles (coacervates) or cell-membrane-like vesicle structures. I believe these findings will allow us to better understand the fundamental release mechanism of AHBs in CFPS toward the development of a life-like autonomous function in protein-assembled vesicles for therapeutic applications.