Isolation and Characterization of Ice-Binding Proteins from Higher Plants

  • Melissa BredowEmail author
  • Heather E. Tomalty
  • Laurie A. Graham
  • Audrey K. Gruneberg
  • Adam J. Middleton
  • Barbara Vanderbeld
  • Peter L. Davies
  • Virginia K. Walker
Part of the Methods in Molecular Biology book series (MIMB, volume 2156)


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.

Key words

Ice-binding proteins Antifreeze proteins Ice-recrystallization inhibition Thermal hysteresis Ice crystals Ice-affinity purification Recombinant protein purification Transgenic IBP expression 



We would like to thank Dr. M. Kuiper, along with many undergraduate students who have participated in data collection and “troubleshooting” these techniques over the years. The research was supported by CIHR and NSERC (Canada) grants to PLD and VKW, respectively. PLD holds the Canada Research Chair in Protein Engineering.


  1. 1.
    Bar DM, Braslavsky I, Davies PL (2016) Ice-binding proteins and their function. Annu Rev Biochem 85:515–542Google Scholar
  2. 2.
    Vrielink ASO, Aloi A, Olijve LLC (2016) Interaction of ice binding proteins with ice, water and ions. Biointerphases II:11Google Scholar
  3. 3.
    Voets IK (2017) From ice-binding proteins to bio-inspired antifreeze materials. Soft Matter 18:4808–4823Google Scholar
  4. 4.
    Bredow M, Walker VK (2017) Ice-binding proteins in plants. Front Plant Sci 8:2153PubMedPubMedCentralGoogle Scholar
  5. 5.
    Duman JG (2015) Animal ice-binding (antifreeze) proteins and glycolipids: an overview with an emphasis on physiological function. J Exp Bot 218:1846–1855Google Scholar
  6. 6.
    Pearce RS (2001) Plant freezing and damage. Ann Bot 87:417–424Google Scholar
  7. 7.
    Knight CA, Wen D, Laursen RA (1995) Nonequilibrium antifreeze peptides and the recrystallization of ice. Cryobiology 32:23–34PubMedGoogle Scholar
  8. 8.
    Sidebottom C, Buckley S, Pudney P et al (2000) Heat-stable antifreeze protein from grass. Nature 406:256PubMedGoogle Scholar
  9. 9.
    Duman JG, Wisniewski MJ (2014) The use of antifreeze proteins for frost protection in sensitive crop plants. Botany 106:60–69Google Scholar
  10. 10.
    Zhang C, Fei S-Z, Arora R et al (2010) Ice recrystallization inhibition proteins of perennial ryegrass enhance freezing tolerance. Planta 232:155–164PubMedGoogle Scholar
  11. 11.
    Bredow M, Vanderbeld B, Walker VK (2016) Ice-binding proteins confer freezing tolerance in transgenic Arabidopsis thaliana. Plant Biotechnol J 15:68–81PubMedPubMedCentralGoogle Scholar
  12. 12.
    Balamurugan S, Ann JS, Varghese IP et al (2018) Heterologous expression of Lolium perenne antifreeze protein confers chilling tolerance in tomato. J Integr Agric 17:1128–1136Google Scholar
  13. 13.
    Ding X, Zhang H, Wang L et al (2014) Effect of barley antifreeze protein on thermal properties and water state of dough during freezing and freeze-thaw cycles. Food Hydrocoll 47:32–40Google Scholar
  14. 14.
    Zhang Y, Zhang H, Ding X et al (2016) Purification and identification of antifreeze protein from cold-acclimated oat (Avena sativa L.) and the cryoprotective activities in ice cream. Food Bioprocess Technol 9:1746Google Scholar
  15. 15.
    Kaleda A, Tsanev R, Klesment T et al (2018) Ice cream structure modification by ice-binding proteins. Food Chem 246:164–171PubMedGoogle Scholar
  16. 16.
    Provesi JG, Neto PAV, Arisi ACM et al (2019) Extraction of antifreeze proteins from cold acclimated leaves of Drimys angustifolia and their application to star fruit (Averrhoa carambola). Food Chem 289:65–73PubMedGoogle Scholar
  17. 17.
    Urrutia ME, Duman JG, Knight CA (1992) Plant thermal hysteresis proteins. Biochim Biophys Acta 1121:199–206PubMedGoogle Scholar
  18. 18.
    Duman JG (1994) Purification and characterization of a thermal hysteresis protein from a plant, the bittersweet nightshade Solanum dulcamara. Biochim Biophys Acta 1206:129–135PubMedGoogle Scholar
  19. 19.
    Hon W-C, Griffith M, Chong P et al (1994) Extraction and isolation of antifreeze proteins from winter rye (Secale cereale L.) leaves. Plant Physiol 104:971–980PubMedPubMedCentralGoogle Scholar
  20. 20.
    Huang T, Duman JG (1995) Purification and characterization of thermal hysteresis protein from cold-acclimated kale, Brassica oleracea. Cryobiology 32:577–581Google Scholar
  21. 21.
    Griffith M, Antikainen M, Hon W-C et al (1997) Antifreeze proteins in winter rye. Physiol Plant 100:327–332Google Scholar
  22. 22.
    Lu CF, Wang H, Jian LC et al (1998) Progress in study of plant antifreeze proteins. Prog Biochem Biophys 25:210–216Google Scholar
  23. 23.
    Hoshino T, Odaira M, Yoshida M et al (1992) Physiological and biochemical significance of antifreeze substances in plants. J Plant Res 112:255–261Google Scholar
  24. 24.
    Worrall D, Elias L, Ashford D et al (1998) A carrot leucine-rich-repeat protein that inhibits ice recrystallization. Science 282:115–117PubMedGoogle Scholar
  25. 25.
    Aticia Ö, Nalbantoǧlu B (2003) Antifreeze proteins in higher plants. Phytochemistry 64:1187–1196Google Scholar
  26. 26.
    Wang W, Wei L, Wang G (2003) Multi-step purification of an antifreeze protein from Ammopiptanthus mongolicus by chromatographic and electrophoretic methods. J Chromatogr Sci 41:489–493PubMedGoogle Scholar
  27. 27.
    Moffatt B, Ewart V, Eastman A (2006) Cold comfort: plant antifreeze proteins. Physiol Plant 126:5–16Google Scholar
  28. 28.
    Zhang C, Zhang H, Wang L et al (2007) Purification of antifreeze protein from wheat bran (Triticum aestivum L.) based on its hydrophilicity and ice-binding capacity. J Agric Food Chem 55:7654–7658PubMedGoogle Scholar
  29. 29.
    Cai Y, Liu S, Liao X et al (2011) Purification and partial characterization of antifreeze proteins from leaves of Ligustrum lucidum Ait. Food Bioprod Process 89:98–102Google Scholar
  30. 30.
    Gupta R, Deswal R (2012) Low temperature stress modulated secretome analysis and purification of antifreeze protein from Hippophae rhamnoides, a Himalayan wonder plant. J Proteome Res 11:2684–2696PubMedGoogle Scholar
  31. 31.
    Ding X, Zhang H, Chen H et al (2015) Extraction, purification and identification of antifreeze proteins from cold-acclimated malting barley (Hordeum vulgare L.). Food Chem 175:74–81PubMedGoogle Scholar
  32. 32.
    Sharma B, Sahoo D, Deswal R (2018) Single-step purification and characterization of antifreeze proteins from lead and berry of a freeze-tolerant shrub seabuckthorn (Hippophae rhamnoides). J Sep Sci 41:3938–3945PubMedGoogle Scholar
  33. 33.
    Li J, Qin R, Xu R et al (2018) Isolation and identification of five cold-induced promoters from Oryza sativa. Planta 247:99–111PubMedGoogle Scholar
  34. 34.
    Lauersen KJ, Brown A, Middleton A et al (2011) Expression and characterization of an antifreeze protein from the perennial rye grass, Lolium perenne. Cryobiology 62:194–201PubMedGoogle Scholar
  35. 35.
    Scotter AJ, Marshall CB, Graham LA et al (2006) The basis for hyperactivity of antifreeze proteins. Cryobiology 53:229–239PubMedGoogle Scholar
  36. 36.
    Takamichi M, Nishimiya Y, Miura A et al (2007) Effect of annealing time of an ice crystal on the activity of type III antifreeze protein. FEBS J 274:6469–6476PubMedGoogle Scholar
  37. 37.
    Bar-Dolev M, Celik Y, Wettlaufer JS et al (2012) New insights into ice growth and melting modifications by antifreeze proteins. J R Soc Interface 9:3249–3259PubMedPubMedCentralGoogle Scholar
  38. 38.
    Qin W, Tyshenko MG, Doucet D et al (2006) Characterization of antifreeze protein gene expression in summer spruce budworm larvae. Insect Biochem Mol Biol 36:210–218PubMedGoogle Scholar
  39. 39.
    Liou Y-C, Daley ME, Graham LA et al (2000) Folding and structural characterization of highly disulfide-bonded beetle antifreeze protein produced in bacteria. Protein Expr Purif 19:148–157PubMedGoogle Scholar
  40. 40.
    Bar M, Bar-Ziv R, Scherf T et al (2006) Efficient production of a folded and functional, highly disulfide-bonded beta-helix antifreeze protein in bacteria. Protein Expr Purif 48:243–252PubMedGoogle Scholar
  41. 41.
    Loughran ST, Wells D (2011) Purification of poly-histidine-tagged proteins. Methods Mol Biol 681:311–335PubMedGoogle Scholar
  42. 42.
    Middleton AJ, Marshall CB, Faucher F et al (2012) Antifreeze protein from freeze-tolerant grass has a beta-roll fold with an irregularly structured ice-binding site. J Mol Biol 416:713–724PubMedGoogle Scholar
  43. 43.
    Bredow M, Vanderbeld B, Walker VK (2016) Knockdown of ice-binding proteins in Brachypodium distachyon demonstrates their role in freeze protection. PLoS One 11:e0167941PubMedPubMedCentralGoogle Scholar
  44. 44.
    Marshall CJ, Basu K, Davies PL (2016) Ice-shell purification of ice-binding proteins. Cryobiology 72:258–263PubMedGoogle Scholar
  45. 45.
    Thalhammer A, Hincha DK, Zuther E (2014) Measuring freezing tolerance: electrolyte leakage and chlorophyll fluorescence assays. Methods Mol Biol 1166:15–24PubMedGoogle Scholar
  46. 46.
    Skinner DZ, Garland-Campbell K (2014) Measuring freezing tolerance: survival and regrowth assays. Methods Mol Biol 1166:7–15PubMedGoogle Scholar
  47. 47.
    Bent A (2006) Arabidopsis thaliana flora dip transformation method. Methods Mol Biol 243:87–104Google Scholar
  48. 48.
    Graham LA, Agrawal P, Oleschuk RD et al (2018) High-capacity ice-recrystallization endpoint assay employing superhydrophobic coatings that is equivalent to the ‘splat’ assay. Cryobiology 81:138–144PubMedGoogle Scholar
  49. 49.
    Graham LA, Walker VK, Davies PL (2000) Developmental and environmental regulation of antifreeze proteins in the mealworm beetle. Eur J Biochem 267:6452–6458PubMedGoogle Scholar
  50. 50.
    Møller HJ, Poulsen JH (2009) Staining of glycoproteins/proteoglycans on SDS gels. In: The protein protocols handbook. Springer Protocols Handbooks. Springer, New York, NY, pp 569–574Google Scholar
  51. 51.
    Jin S, Song YN, Deng WY et al (1993) The regulatory VirA protein of Agrobacterium tumefaciens does not function at elevated temperatures. J Bacteriol 175:6830–6835PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Melissa Bredow
    • 1
    Email author
  • Heather E. Tomalty
    • 2
  • Laurie A. Graham
    • 2
  • Audrey K. Gruneberg
    • 2
  • Adam J. Middleton
    • 1
    • 3
  • Barbara Vanderbeld
    • 1
  • Peter L. Davies
    • 1
    • 2
  • Virginia K. Walker
    • 1
    • 2
  1. 1.Department of BiologyQueen’s UniversityKingstonCanada
  2. 2.Department of Biomedical and Molecular SciencesQueen’s UniversityKingstonCanada
  3. 3.Department of BiochemistryUniversity of OtagoDunedinNew Zealand

Personalised recommendations