The rational design and applications of synthetic antifreeze protein mimics

• Main Author: Mitchell, Daniel E.
• Published: University of Warwick 2015
• Subjects: 572; QR Microbiology
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.682906

Antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) are proteins found in a wide variety of organisms adapted to survive in freezing temperatures. These proteins are powerful ice recrystallization inhibitors (IRI), slowing or even stopping ice crystal growth. This property is of significant interest in the area of cryopreservation due to ice recrystallization upon thawing being the limiting factor in the recovery of many biological materials. The need for reliable cryopreservation is becoming more and more necessary in many clinical areas, including regenerative and transplant medicine, and protein based therapeutics. The identification of new protein structures and sequences is crucially to understanding this ability. To this end l-type lectins, which show sequential homology to type 2 AFPs were explored and found to possess calcium dependant IRI activity. In addition, a secondary motif, amphipathicity was investigated. As many AFPs possess separate well defined hydrophilic/ hydrophobic domains this was thought to be a property of high importance. The antimicrobial peptide nisin A, which is amphipathic, was found to also possess cation dependant IRI activity. This identification demonstrates a new approach to identifying IRI active molecules and may further enhance our understanding of the mechanisms involved. As current “gold standard” methods for identification of IRI activity are relatively slow and time consuming signifying that the discovery of new IRI molecules is a slow process. In order to increase throughput of the discovery of novel IRI active molecules a gold nanoparticle assay was developed. Using this method serum proteins were discovered to possess weak but definite activity at higher concentrations. This is of particular interest as serum proteins such as fetal calf serum are commonly used in cryopreservation applications. The isolation of these remarkable proteins from primary sources in significant quantities is difficult and not financially viable, while recombinant expression is complicated by glycosylation of AFGPs. Furthermore non-native proteins may cause immunogenic problems. Therefore synthetic mimetic such as polymers and small molecules are highly appealing. Using a biomimetic approach, amphipathic metallohelicies with alpha helical character similar to that of a type 1 AFP were evaluated for ice recrystallization inhibition. It was found that these metallohelices could be optimized to completely inhibit ice recrystallization, demonstrating that by creating materials which mimic AFPs similar properties can be engineered successfully. In addition, polyampholyte polymers (polymers possessing both positive and negative side chains) were synthesized, and demonstrated to possess IRI activity when the ratio of positive to negative side chains was roughly 1:1. These were then used to significantly enhance post-thaw recovery rates of red blood cells (RBCs). Polymers are appealing as they are highly tunable and can be used to mimic proteins in a wide variety of biological areas. Polyampholyte polymers and previously identified IRI polymer; polyvinyl alcohol (PVA), added to polyethylene glycol were found to be a highly efficient way of preserving proteins and antibodies under freezing conditions. The application of polyampholytes and other IRI active molecules are therefore highly attractive in the cryopreservation of a whole range of biological materials, potentially yielding widespread clinical benefits.