Chilling injury in human kidney tubule cells after subzero storage is not mitigated by antifreeze protein addition.

By preventing freezing, antifreeze proteins (AFPs) can permit cells and organs to be stored at subzero temperatures. As metabolic rates decrease with decreasing temperature, subzero static cold storage (SZ-SCS) could provide more time for tissue matching and potentially lead to fewer discarded organs. Human kidneys are generally stored for under 24 h and the tubule epithelium is known to be particularly sensitive to static cold storage (SCS). Here, telomerase-immortalized proximal-tubule epithelial cells from humans, which closely resemble their progenitors, were used as a proxy to assess the potential benefit of SZ-SCS for kidneys. The effects of hyperactive AFPs from a beetle and Cryostasis Storage Solution were compared to University of Wisconsin Solution at standard SCS temperatures (4 °C) and at -6 °C for up to six days. Although the AFPs helped guard against freezing, lower storage temperatures under these conditions were not beneficial. Compared to cells at 4 °C, those stored at -6 °C showed decreased viability as well as increased lactate dehydrogenase release and apoptosis. This suggests that this kidney cell type might be prone to chilling injury and that the addition of AFPs to enable SZ-SCS may not be effective for increasing storage times.

Isolation and Characterization of Ice-Binding Proteins from Higher Plants.

The characterization of ice-binding proteins (IBPs) from plants can involve many techniques, a few of which are presented here. Chief among these methods are tests for ice recrystallization inhibition, an activity characteristic of plant IBPs. Two related procedures are described, both of which can be used to demonstrate and quantify ice-binding activity. First, is the traditional "splat" assay, which can easily be set up using common laboratory equipment, and second, is our modification of this method using superhydrophobic coated sapphire for analysis of multiple samples in tandem. Thermal hysteresis is described as another method for quantifying ice-binding activity, during which ice crystal morphology observations can be used to provide clues about ice-plane binding. Once ice-binding activity has been evaluated, it is necessary to verify IBP identity. We detail two methods for enriching IBPs from complex mixtures using ice-affinity purification, the "ice-finger" and "ice-shell" methods, and we highlight their advantages and limitations for the isolation of plant IBPs. Recombinant IBP expression, necessary for detailed ice-binding analysis, can present challenges. Here, a strategy for recovery of soluble, active protein is described. Lastly, verification of function in planta borrows from standard protocols, but with an additional screen applicable to IBPs. Together, these methods, and a few considerations critical to success, can be used to assist researchers wishing to isolate and characterize IBPs from plants.

Laboratory-Scale Isolation of Insect Antifreeze Protein for Cryobiology.

Micromolar concentrations of hyperactive antifreeze proteins (AFPs) from insects can prevent aqueous solutions from freezing down to at least -6 °C. To explore cryopreservation of cells, tissues and organs at these temperatures without ice formation, we have developed a protocol to reliably produce ultrapure Tenebrio molitor AFP from cold-acclimated beetle larvae reared in the laboratory. The AFP was prepared from crude larval homogenates through five cycles of rotary ice-affinity purification, which can be completed in one day. Recovery of the AFP at each step was >90% and no impurities were detected in the final product. The AFP is a mixture of isoforms that are more active in combination than any one single component. Toxicity testing of the purified AFP in cell culture showed no inhibition of cell growth. The production process can easily be scaled up to industrial levels, and the AFP used in cryobiology applications was recovered for reuse in good yield and with full activity.

Ice and anti-nucleating activities of an ice-binding protein from the annual grass, Brachypodium distachyon.

Plants exposed to sub-zero temperatures face unique challenges that threaten their survival. The growth of ice crystals in the extracellular space can cause cellular dehydration, plasma membrane rupture and eventual cell death. Additionally, some pathogenic bacteria cause tissue damage by initiating ice crystal growth at high sub-zero temperatures through the use of ice-nucleating proteins (INPs), presumably to access nutrients from lysed cells. An annual species of brome grass, Brachypodium distachyon (Bd), produces an ice-binding protein (IBP) that shapes ice with a modest depression of the freezing point (~0.1 °C at 1 mg/mL), but high ice-recrystallization inhibition (IRI) activity, allowing ice crystals to remain small at near melting temperatures. This IBP, known as BdIRI, is unlike other characterized IBPs with a single ice-binding face, as mutational analysis indicates that BdIRI adsorbs to ice on two faces. BdIRI also dramatically attenuates the nucleation of ice by bacterial INPs (up to -2.26 °C). This 'anti-nucleating' activity is significantly higher than previously documented for any IBP.

Identification of Plant Ice-binding Proteins Through Assessment of Ice-recrystallization Inhibition and Isolation Using Ice-affinity Purification.

Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In plants, freeze damage occurs when extracellular ice crystals grow, resulting in the rupture of plasma membranes and possible cell death. Adsorption of IBPs to ice crystals restricts further growth by a process known as ice-recrystallization inhibition (IRI), thereby reducing cellular damage. IBPs also demonstrate the ability to depress the freezing point of a solution below the equilibrium melting point, a property known as thermal hysteresis (TH) activity. These protective properties have raised interest in the identification of novel IBPs due to their potential use in industrial, medical and agricultural applications. This paper describes the identification of plant IBPs through 1) the induction and extraction of IBPs in plant tissue, 2) the screening of extracts for IRI activity, and 3) the isolation and purification of IBPs. Following the induction of IBPs by low temperature exposure, extracts are tested for IRI activity using a 'splat assay', which allows the observation of ice crystal growth using a standard light microscope. This assay requires a low protein concentration and generates results that are quickly obtained and easily interpreted, providing an initial screen for ice binding activity. IBPs can then be isolated from contaminating proteins by utilizing the property of IBPs to adsorb to ice, through a technique called 'ice-affinity purification'. Using cell lysates collected from plant extracts, an ice hemisphere can be slowly grown on a brass probe. This incorporates IBPs into the crystalline structure of the polycrystalline ice. Requiring no a priori biochemical or structural knowledge of the IBP, this method allows for recovery of active protein. Ice-purified protein fractions can be used for downstream applications including the identification of peptide sequences by mass spectrometry and the biochemical analysis of native proteins.

Perturbation of bacterial ice nucleation activity by a grass antifreeze protein.

Certain plant-associating bacteria produce ice nucleation proteins (INPs) which allow the crystallization of water at high subzero temperatures. Many of these microbes are considered plant pathogens since the formed ice can damage tissues, allowing access to nutrients. Intriguingly, certain plants that host these bacteria synthesize antifreeze proteins (AFPs). Once freezing has occurred, plant AFPs likely function to inhibit the growth of large damaging ice crystals. However, we postulated that such AFPs might also serve as defensive mechanisms against bacterial-mediated ice nucleation. Recombinant AFP derived from the perennial ryegrass Lolium perenne (LpAFP) was combined with INP preparations originating from the grass epiphyte, Pseudomonas syringae. The presence of INPs had no effect on AFP activity, including thermal hysteresis and ice recrystallization inhibition. Strikingly, the ice nucleation point of the INP was depressed up to 1.9°C in the presence of LpAFP, but a recombinant fish AFP did not lower the INP-imposed freezing point. Assays with mutant LpAFPs and the visualization of bacterially-displayed fluorescent plant AFP suggest that INP and LpAFP can interact. Thus, we postulate that in addition to controlling ice growth, plant AFPs may also function as a defensive strategy against the damaging effects of ice-nucleating bacteria.

Isolation and characterization of ice-binding proteins from higher plants.

The characterization of ice-binding proteins from plants can involve many techniques, only a few of which are presented here. Chief among these methods are tests for ice recrystallization inhibition activity. Two distinct procedures are described; neither is normally used for precise quantitative assays. Thermal hysteresis assays are used for quantitative studies but are also useful for ice crystal morphologies, which are important for the understanding of ice-plane binding. Once the sequence of interest is cloned, recombinant expression, necessary to verify ice-binding protein identity can present challenges, and a strategy for recovery of soluble, active protein is described. Lastly, verification of function in planta borrows from standard protocols, but with an additional screen applicable to ice-binding proteins. Here we have attempted to assist researchers wishing to isolate and characterize ice-binding proteins from plants with a few methods critical to success.