Falling water ice affinity purification of ice-binding proteins

Adar, Chen; Sirotinskaya, Vera; Dolev, Maya Bar; Friehmann, Tomer; Braslavsky, Ido

doi:10.1038/s41598-018-29312-x

Cited by 20 publications

(18 citation statements)

References 36 publications

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“…amenable to biotechnology applications. However, the lack of a conserved IBP domain makes finding new examples labour‐intensive, requiring the blind sampling of different species for activity followed by native purification of the protein for identification . DUF3494 IBPs are an easy‐to‐find alternative, detectable through simple blast searches, allowing for recombinant expression for characterization.…”

Section: Discussionmentioning

confidence: 99%

Ice‐binding proteins and the ‘domain of unknown function’ 3494 family

et al. 2019

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Ice‐binding proteins (IBPs) control the growth and shape of ice crystals to cope with subzero temperatures in psychrophilic and freeze‐tolerant organisms. Recently, numerous proteins containing the domain of unknown function (DUF) 3494 were found to bind ice crystals and, hence, are classified as IBPs. DUF3494 IBPs constitute today the most widespread of the known IBP families. They can be found in different organisms including bacteria, yeasts and microalgae, supporting the hypothesis of horizontal transfer of its gene. Although the 3D structure is always a discontinuous β‐solenoid with a triangular cross‐section and an adjacent alpha‐helix, DUF3494 IBPs present very diverse activities in terms of the magnitude of their thermal hysteresis and inhibition of ice recrystallization. The proteins are secreted into the environments around the host cells or are anchored on their cell membranes. This review covers several aspects of this new class of IBPs, which promise to leave their mark on several research fields including structural biology, protein biochemistry and cryobiology.

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Section: Discussionmentioning

confidence: 99%

Ice‐binding proteins and the ‘domain of unknown function’ 3494 family

et al. 2019

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“…The IAP procedure can be scaled to a much larger vessel size with multiple units simultaneously operating. Alternatively, the falling water ice-affinity purification method that uses commercial ice making machines can be employed [25]. These procedures might be sufficient to produce food-grade Tm AFP, especially since T. molitor larvae are an accepted food source and are rich in protein [26].…”

Section: Discussionmentioning

confidence: 99%

Laboratory-Scale Isolation of Insect Antifreeze Protein for Cryobiology

et al. 2019

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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.

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“…Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted in a 6% stacking gel and a 16% solving gel following a previously-used procedure [20]. Mass spectrometry analysis was performed on a Microflex LT mass spectrometer (Bruker Daltonics, Billerica, MA, USA) using FlexControl software.…”

Section: Methodsmentioning

confidence: 99%

Ice Nucleation Properties of Ice-binding Proteins from Snow Fleas

et al. 2019

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Ice-binding proteins (IBPs) are found in many organisms, such as fish and hexapods, plants, and bacteria that need to cope with low temperatures. Ice nucleation and thermal hysteresis are two attributes of IBPs. While ice nucleation is promoted by large proteins, known as ice nucleating proteins, the smaller IBPs, referred to as antifreeze proteins (AFPs), inhibit the growth of ice crystals by up to several degrees below the melting point, resulting in a thermal hysteresis (TH) gap between melting and ice growth. Recently, we showed that the nucleation capacity of two types of IBPs corresponds to their size, in agreement with classical nucleation theory. Here, we expand this finding to additional IBPs that we isolated from snow fleas (the arthropod Collembola), collected in northern Israel. Chemical analyses using circular dichroism and Fourier-transform infrared spectroscopy data suggest that these IBPs have a similar structure to a previously reported snow flea antifreeze protein. Further experiments reveal that the ice-shell purified proteins have hyperactive antifreeze properties, as determined by nanoliter osmometry, and also exhibit low ice-nucleation activity in accordance with their size.

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Falling water ice affinity purification of ice-binding proteins

Cited by 20 publications

References 36 publications

Ice‐binding proteins and the ‘domain of unknown function’ 3494 family

Ice‐binding proteins and the ‘domain of unknown function’ 3494 family

Laboratory-Scale Isolation of Insect Antifreeze Protein for Cryobiology

Ice Nucleation Properties of Ice-binding Proteins from Snow Fleas

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