Master of Science (MS)
Semester of Degree Completion
Gary A. Bulla
Anhydrobiosis is an astounding strategy that allows certain species (both animals and plants) to survive severe environmental conditions such as desiccation, extreme cold, or heat in the habitat. Despite the occurrence of several different molecular strategies, expression of highly hydrophilic polypeptides termed LEA proteins has been most conclusive identified as a requirement for the survival of plants and animals during periods of water stress such as freezing and drying. Several classification schemes for LEA proteins have been proposed and the brine shrimp, Artemia franciscana, is the only known animal that naturally expresses LEA proteins from three different classification groups (groups 1, 3, and 6). LEA proteins occur in different subcellular compartments including the cytosol and mitochondria. To understand the biochemical properties of LEA proteins, it is important to characterize their structure. LEA proteins are intrinsically disordered in aqueous solution and the exact structure and function of these proteins in the dry and/or hydrated states is still poorly defined and understood. We found, that a purified group 1 LEA protein from A. franciscana (AfrLEA 1.1) helped to retain enzyme activity after desiccation of lactate dehydrogenase (LDH) for land 7 days in the presence or absence of BSA or trehalose or other purified LEA protein. Increased concentration of purified AfrLEA 1.1, increased the percentage of LDH activity retained after desiccation. To further characterize AfrLEA 1.1, we cloned, expressed, and purified the protein in E. coli. We purified untagged AfrLEA 1.1 protein by affinity chromatography via Intein Mediated Purification with an Affinity Chitin-binding Tag system; a novel protein purification system which utilizes the inducible self-cleavage activity of protein splicing elements (termed inteins) to separate the target protein from the affinity tag. Furthermore, AfrLEA1.1 was expressed in Nicotiana tabacum to investigate if the protein increases drought tolerance of this model plant. Tobacco plants with confirmed transgenic AfrLEA1.1 were subjected to water stress in the presence of polyethylene glycol (PEG; 10,000 MW) at increasing percentages to investigate the impact of osmotic stress on plant survival. PEG-stressed transgenic LEA plants showed significantly faster growth of roots compared to non-transgenic GUS control plants under the same conditions both if measured as an increase in fresh weight (P=0.033, P<0.05) or dry weight (P=0.028, P<0.05). This result clearly indicates a better capability to cope with water stress in presence of AfrLEA1.1 and points to a function of this protein not only during desiccation but also under less severe osmotic stress conditions. Transgenic LEA plants also showed a significantly increased level of total growth compared to controls, measured as an increase in total fresh weight (P=0.0461, P<0.05) and total dry weight (P=0.0342, P<0.05) under standard growth conditions. Along with the better growth of roots under osmotic stress condition and better overall somatic growth under control condition, they also showed a significantly higher amount of chlorophyll content after freezing condition compares to room temperature.
Karim, Md Fazlul, "Expression, Purification, and Characterization of an Intrinsically Disordered Late Embryogenesis Abundant (LEA) Protein from Artemia franciscana Utilizing Escherichia coli and Nicotiana tabacum" (2017). Masters Theses. 2862.