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.

[1]  I. Braslavsky,et al.  Falling water ice affinity purification of ice-binding proteins , 2018, Scientific Reports.

[2]  T. Vance,et al.  An ice‐binding and tandem beta‐sandwich domain‐containing protein in Shewanella frigidimarina is a potential new type of ice adhesin , 2018, The FEBS journal.

[3]  A. Vilcinskas,et al.  Sustainable farming of the mealworm Tenebrio molitor for the production of food and feed , 2017, Zeitschrift fur Naturforschung. C, Journal of biosciences.

[4]  V. Walker,et al.  Kidney preservation at subzero temperatures using a novel storage solution and insect ice-binding proteins. , 2017, Cryo letters.

[5]  P. Davies,et al.  Ice-shell purification of ice-binding proteins. , 2016, Cryobiology.

[6]  P. Davies.  Ice-binding proteins: a remarkable diversity of structures for stopping and starting ice growth. , 2014, Trends in biochemical sciences.

[7]  J. Duman,et al.  Recombinant Dendroides canadensis antifreeze proteins as potential ingredients in cryopreservation solutions. , 2014, Cryobiology.

[8]  Christopher B. Marshall,et al.  Antifreeze protein from freeze-tolerant grass has a beta-roll fold with an irregularly structured ice-binding site. , 2012, Journal of molecular biology.

[9]  M. Brockbank,et al.  Lessons from nature for preservation of mammalian cells, tissues, and organs , 2011, In Vitro Cellular & Developmental Biology - Animal.

[10]  I. Eleftherianos,et al.  Role and Importance of Phenoloxidase in Insect Hemostasis , 2010, Journal of Innate Immunity.

[11]  P. Davies,et al.  Direct visualization of spruce budworm antifreeze protein interacting with ice crystals: basal plane affinity confers hyperactivity. , 2008, Biophysical journal.

[12]  R. Bar-Ziv,et al.  Efficient production of a folded and functional, highly disulfide-bonded beta-helix antifreeze protein in bacteria. , 2006, Protein expression and purification.

[13]  P. Davies,et al.  Challenges in the expression of disulfide bonded, threonine-rich antifreeze proteins in bacteria and yeast. , 2006, Protein expression and purification.

[14]  J. Duman,et al.  Antifreeze proteins of the beetle Dendroides canadensis enhance one another's activities. , 2005, Biochemistry.

[15]  C. Hew,et al.  Liver-specific and seasonal expression of transgenic Atlantic salmon harboring the winter flounder antifreeze protein gene , 2004, Transgenic Research.

[16]  P. Davies,et al.  Purification of antifreeze proteins by adsorption to ice. , 2003, Biochemical and biophysical research communications.

[17]  B. Sykes,et al.  Identification of the ice‐binding face of antifreeze protein from Tenebrio molitor , 2002, FEBS letters.

[18]  J. Duman,et al.  Antifreeze and ice nucleator proteins in terrestrial arthropods. , 2001, Annual review of physiology.

[19]  P. Davies,et al.  Developmental and environmental regulation of antifreeze proteins in the mealworm beetle Tenebrio molitor. , 2000, European journal of biochemistry.

[20]  Michael J. Kuiper,et al.  β-Helix structure and ice-binding properties of a hyperactive antifreeze protein from an insect , 2000, Nature.

[21]  B. Sykes,et al.  Folding and structural characterization of highly disulfide-bonded beetle antifreeze protein produced in bacteria. , 2000, Protein expression and purification.

[22]  P. Thibault,et al.  A complex family of highly heterogeneous and internally repetitive hyperactive antifreeze proteins from the beetle Tenebrio molitor. , 1999, Biochemistry.

[23]  Y. Liou,et al.  Hyperactive antifreeze protein from beetles , 1997, Nature.

[24]  T. Mosmann.  Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. , 1983, Journal of immunological methods.

[25]  J. Raymond,et al.  Adsorption inhibition as a mechanism of freezing resistance in polar fishes. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[26]  Donald E. Wohlschlag,et al.  Freezing Resistance in Some Antarctic Fishes , 1969, Science.