Overcoming bottlenecks for in vitro synthesis and initial structural insight of ice nucleating protein InaZ

Unlike inorganic or other synthetic alternatives, ice nucleating proteins (INPs) remain the most efficient ice nuclei today. Their potential applications in cryo-preservation, biomedicine, food industry and in the modulation of climate are widespread. Nevertheless, over several decades, cell-based recombinant methods have experienced multiple difficulties expressing these large proteins in full-length and in necessary yields while retaining functionality. As a result, our understanding of the structure and ice nucleation mechanism for this class of proteins is incomplete, and, most importantly, the full extent of possible applications unrealized. Using a wheat-germ cell-free expression pipeline, we successfully expressed and purified full-length ice nucleating protein InaZ from Pseudomonas syringae , known as a model INP. High protein yield and solubility has been achieved using this system. Ice nucleation experiments inside a dynamic environmental scanning electron microscope (ESEM) confirmed that the produced InaZ products remain functional. Preliminary structural assessments of these proteins using Transmission Electron Microscopy (TEM) showed experimental evidence for their structural organization as fibrils. We believe that the current platform will be suitable for expressing other INPs of interest and can be further employed as new engineering system either for industrial or scientific needs.