Regulating Protein Immobilization During Cell-Free Protein Synthesis in Hyaluronan Microgels

Cell-like platforms are being studied intensively for their application in synthetic biology to mimic aspects of life in an artificial environment. Here, micrometer-sized, bifunctional microgels are used as an experimental platform to investigate the interplay of cell-free protein synthesis (CFPS) and in situ protein accumulation inside the microgel volume. In detail, microgels made of hyaluronic acid (HA) are first modified with different amounts of nitrilotriacetic acid (NTA) moieties to characterize the capability and maximum capacity of binding His-tag modified GFP. CFPS is optimized for the system used here, particularly when using a linear DNA template. Afterward, HA-microgels are functionalized with the linear DNA template and Ni²⁺-activated NTA moieties to bind in situ synthesized GFP-His. CFPS and parallel protein accumulation within the microgels are observed over time to determine the GFP-His binding to the microgel platform. With this approach, the study presents the first steps for a platform to study the temporal-spatial regulation of protein synthesis by tailored protein binding or release from the microgel matrix-based reaction environment.