Graduate Program

Biological Sciences

Degree Name

Master of Science (MS)

Semester of Degree Completion

2013

Thesis Director

Michael A. Menze

Thesis Committee Member

Gary A. Bulla

Thesis Committee Member

Janice Coons

Abstract

Animals possessing tolerance to extreme water stress are termed anhydrobiotes. Many desiccation tolerant organisms respond to water stress by intracellular accumulation of selected sugars such as trehalose and larger macromolecules such as Late Embryogenesis Abundant (LEA) proteins and thereby maintain the cell viability. Evidence indicates that the presence of trehalose and Late Embryogenesis Abundant (LEA) proteins may work synergistically to confer cellular protection during drying in eukaryotic cells. We evaluated any increase in cellular desiccation tolerance by expressing different LEA proteins in a non-desiccation tolerant insect cell line Drosophila melanogaster (fruit fly) (Kc167 cells) in the presence of trehalose.

Transgenic group I LEA protein expressing cells (Kc167- LEA 1.1 and Kc167- LEA 1.3) and group V LEA protein expressing cells (Kc167- LEA 5) were used for convective droplet drying experiments. Experiments were carried out with and without 200 mM extracellular trehalose. Statistical analysis demonstrated that there was no significant difference (ANCOVA: F1, 171.13 = 37.99, p =0.5217, r2 = 0.76) between the amount of viable cells after desiccation of Kc167 control and LEA 1.3 expressing cells in the absence of extracellular trehalose. Interestingly, After adding extracellular trehalose to the culture medium, Kc167-LEA 1.3 cells showed significant increase in viability after desiccation compared to the control cells (ANCOVA: F1, 182.06 = 39.20, p < 0.0001, r2 = 0.79). Similarly, Kc167-LEA 1.1 showed a significant increase in the viable cells compared to the control Kc167 cells after desiccation. (ANCOVA: F1, 125.03= 41.04, p < 0.0001, r2 = 0.80). Transgenic expression of LEA 5 protein also showed significantly increased cell viability of Kc167 cells during droplet drying in presence of 200 mM trehalose compared to control Kc167 cells (ANCOVA: F1, 153.10 = 39.24, p < 0.0001, r2 = 0.77).

To investigate the properties of LEA protein on protection of macromolecular proteins such as enzymes at sub-freezing temperatures, lactate dehydrogenase enzyme assay was carried out. Purified LEA 1.1 protein (0.050 mg/ml) was able to protect lactate dehydrogenase enzyme activity 10 h after freezing at -20°C (n = 3, ±SE, P < 0.05). Surprisingly, cryomicroscope results have showed that addition of 0.050 mg/ml LEA 1.1 protein into 40 mM HEPES buffer have reduced the freezing temperature of 40 mM HEPES from -14.4°C to -17.3°C changing the ice-crystal shape from rectangular to pentagonal shape. Moreover, protein foldlndex of Kyte-Doolittle plot was performed for group I LEA proteins (LEA 1.1 and LEA 1.3) and showed that both these proteins are unfolded at its native stage while the LEA 5 protein is likely folded protein in aqueous solution.

These results suggest that trehalose and late embryogenesis abundant (LEA) proteins work synergistically to confer cellular protection during insect cell desiccation enlightening the potential of insect cell storage without cryopreservation. Moreover, LEA proteins can be utilized as a potential cryoprotecting agent to minimize the freezing damage of biomolecules that has to be cryopreserved.

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