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Isolation and Characterization of Antifreeze Proteins from the Antarctic Marine Microalga Pyramimonas gelidicola

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Abstract

Antifreeze proteins (AFPs) play an important role in the psychrophilic adaptation of polar organisms. AFPs encoded by an Antarctic chlorophyte, identified as Pyramimonas gelidicola, were isolated and characterized. Two AFP isoforms were found from cDNAs and their deduced molecular weights were estimated to be 26.4 kDa (Pg-1-AFP) and 27.1 kDa (Pg-2-AFP). Both AFP cDNAs were cloned and expressed in Escherichia coli. The purified recombinant Pg-1-rAFP and Pg-2-rAFP both showed antifreeze activity based on the measurement of thermal hysteresis (TH) and morphological changes to single ice crystals. Pg-1-rAFP shaped ice crystals into a snowflake pattern with a TH value of 0.6 ± 0.02 °C at ~15 mg/ml. Single ice crystals in Pg-2-rAFP showed a dendritic morphology with a TH value of 0.25 ± 0.02 °C at the same protein concentration. Based on in silico protein structure predictions, the three-dimensional structures of P. gelidicola AFPs match those of their homologs found in fungi and bacteria. They fold as a right-handed β-helix flanked by an α-helix. Unlike the hyperactive insect AFPs, the proposed ice-binding site on one of the flat β-helical surfaces is neither regular nor well-conserved. This might be a characteristic of AFPs used for freeze tolerance as opposed to freeze avoidance. A role for P. gelidicola AFPs in freeze tolerance is also consistent with their relatively low TH values.

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Acknowledgements

This work was supported by the National Agenda Project from the Korea Research Council of Fundamental Science and Technology (KRCF) and the Korea Polar Research Institute (KOPRI) (Grant No. PG12010). This work was supported by a Korea CCS R&D Center (KCRC) grant funded by the Korean government (Ministry of Science, ICT and Future Planning). PLD holds a Canada Research Chair in Protein Engineering and acknowledges research support from the Canadian Institutes for Health Research.

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Authors and Affiliations

  1. Department of Life Science, Division of Natural Sciences, Hanyang University, 133-791, Seoul, South Korea

    Woongsic Jung, Yunho Gwak & EonSeon Jin

  2. Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, ON, Canada, K7L 3N6

    Peter L. Davies

  3. Department of Chemistry, Division of Natural Sciences, Pukyong National University, 608-737, Busan, South Korea

    Hak Jun Kim

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  1. Woongsic Jung
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  2. Yunho Gwak
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Correspondence to Hak Jun Kim or EonSeon Jin.

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Fig. S1

Information on amino acid sequences of Pg-AFPs including the signal peptides. Signal peptides are predicted by SignalP v4.0 (Peterson et al. 2011) and indicated by bold, underlined residues. Asterisks indicate stop codons (DOCX 17 kb)

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Jung, W., Gwak, Y., Davies, P.L. et al. Isolation and Characterization of Antifreeze Proteins from the Antarctic Marine Microalga Pyramimonas gelidicola . Mar Biotechnol 16, 502–512 (2014). https://doi.org/10.1007/s10126-014-9567-y

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