A number of different proteins have evolved to interfere with ice crystal growth. These are known as antifreeze proteins (AFPs), thermal hysteresis proteins, ice structuring proteins, or ice binding proteins. Antifreeze proteins inhibit ice recrystallization and nucleation, modify ice morphology, and display thermal hysteresis – the depression of the freezing point of water without altering the melting point (Bar et al., 2006). The activity of antifreeze proteins varies from about 1oC depression of freezing temperature in fish to 5-7oC depression in the hemolymph of many overwintering insects (Strom et al., 2006). Antifreeze proteins have a variety of applications in cryopreservation of food, cells, and organs, as well as in cryosurgery, agriculture, and as non-polluting de-icing agents. These compounds are more than 300 times more effective in preventing freezing than conventional chemical antifreezes at the same concentrations (Graham, 1997).
While moderately active fish AFPs are already being used in industry (Unilever and Breyers, for example incorporate fish AFPs into some of their American ice creams to allow for production of very creamy, dense, reduced fat ice cream with fewer additives), to the best of our knowledge, hyperactive AFPs have not been utilized for applications outside of basic science. This may seem surprising at first, since the Tenebrio molitor antifreeze protein has up to 100 times the specific activity of fish antifreeze proteins (Graham, 1997). One of the main reasons for the lack of use of hyperactive antifreeze proteins in industry is that production of these proteins is prohibitively expensive and currently inefficient compared to moderate fish AFPs. Recombinant expression of most hyperactive antifreeze proteins results in inclusion bodies of largely inactive material and requires laborious and expensive refolding protocols.
Recent studies have isolated a novel, hyperactive antifreeze protein from the hemolymph of the cold tolerant Siberian cerambycid beetle, Rhagium inquisitor (RiAFP) (Kristiansen, 2011). The only reported details of this protein thus far are its primary sequence, and its thermal hysteresis activity. Thermal hysteresis measurements indicate that RiAFP is the most active AFP isolated thus far. The protein seems to have significantly fewer disulfide bonds and fewer repeated sequences compared to other active insect AFPs. Whereas the Tenebrio molitor antifreeze protein has a cysteine content of 19% and a total of eight disulfide bonds in its core, the Rhagium Inquisitor AFP has a cysteine content of <1% and has only one disulfide bond (Kristiansen, 2011). These features, together with its small size (12kDa), lead our team to hypothesize that RiAFP would make a good candidate for recombinant expression. Our hope is to demonstrate that RiAFP is an attractive reagent for applications requiring freeze resistance or the control of ice growth and morphology. Importantly, we provide some of the first functional characterizations of this protein, since little is known about its structure or activity other than its thermal hysteresis properties.
Recently, multiplex automated genome engineering (MAGE) was developed for large-scale programming and evolution of cells (Wang, 2009). MAGE allows rapid generation of sequence diversity across a large population of cells through oligo-mediated allelic replacement (Wang, 2009). Synthetic oligonucleotides are repeatedly and continuously introduced to a population of cells and are incorporated at the lagging strand of the replication fork during DNA replication. This approach was previously used to optimize the DXP biosynthesis pathway in E coli to overproduce the industrially important isoprenoid lycopene.
We designed degenerate oligos to target several sites of the hypothesized ice-binding pocket of the protein. The hypothesized binding site of RiAFP contains stretches riches in alternating Thr and Ala residues, similar to nonhomolgous Thr-rich AFPs. The spacing between ice-binding Thr residues is a close match to the spacing of oxygen atoms on several planes of ice. Twenty two sets of degenerate oligos were designed to insert additional Tx repeats, delete Tx repeats, delete entire TxT segments, and replace regions with degenerate TxTxTxT repeats. After incorporating RiAFP into the genome of the EcNR2 strain (using labda-red recombination) MAGE was used to generate a diverse population of mutants for the antifreeze protein sequence. Multiple freeze thaw cycles were applied as a selective pressure for enhanced survivability after each MAGE cycle.
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