Falling Water Ice Purification (FWIP): a New Method for Large Scale Purification of Ice-Binding Proteins

Chen Adar chen.adar@mail.huji.ac.il Vera Sirotinskaya Maya Bar Dolev Tomer Friehmann Ido Braslavsky
Institute of Biochemistry, Food Science and Nutrition, The Robert H. Smith Faculty of Agriculture, Food, and Environment, The Hebrew University of Jerusalem, Rehovot, Israel

Ice Binding Proteins (IBPs) permit various organisms` survival at subzero environments. The utilization of IBPs allows their hosts to thrive in the presence of ice, conditions that can otherwise be lethal. The potential of IBPs as additives in various industries ranging from food storage, cryopreservation, anti-icing and slurry systems is evident. This potential is far from realization due to lack of sufficient availability and high cost of production of high quality IBPs. The main objective of this research is to reach this gap by developing a novel method of large scale purification of IBPs. Our method, named Falling Water Ice Purification (FWIP), is based on affinity of IBPs to ice. Fed-batch fermentation is used to obtain high-density cultures of E. coli expressing recombinant IBPs. The cell lysate containing IBPs is allowed to flow over a 900 cm2 chilled vertical surface. The temperature of this surface is lowered over the course of 40 minutes, and upon sufficient cooling, ice crystals with vast surface area are produced. IBPs adsorb to ice crystals while non IBPs solutes are excluded. We observed a correlation between IBPs concentration in the running solution and the IBPs concentration in ice fraction. By using FWIP we purified IBPs (e.g. Type-III AFP, eGFP-RiAFP, TmAFP and MBP-TmAFP), up to 250 mg in the case of Type-III AFP, which indicates that FWIP is suitable for large scale IBP purification (hundreds of miligrams/day). After two purification rounds with FWIP we got >95% purity of IBPs. This method opens possibilities for IBP-fused compounds (e.g. anti-bodies, enzymes, bio-materials, etc.) purification, as well as wild type IBP purification without affinity tags.

We acknowledge funding by ISF and ERC.









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