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Expression, purification, and characterization of an intrinsically disordered Late Embryogenesis Abundant (LEA) protein from Artemia franciscana utilizing Escherichia coli and Nicotiana tabacum
Author(s) -
Karim Md Fazlul,
Yordanov Yordan S.,
Menze Michael A.
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.914.3
Subject(s) - cryptobiosis , desiccation , biology , desiccation tolerance , biochemistry , enzyme , lactate dehydrogenase , trehalose , escherichia coli , brine shrimp , botany , gene , ecology
Anhydrobiosis is an astounding strategy that allows certain species (both in animals and plants) to survive severe environmental conditions such as desiccation, extreme cold, or heat in the habitat. Among different molecular strategies, expression of highly hydrophilic polypeptides termed LEA proteins has been linked to 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 several subcellular compartments including the cytosol and mitochondria. To investigate 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 is still poorly defined and understood. We hypothesized that LEA proteins will show different protective properties depending on the target enzyme (e.g. lactate dehydrogenase vs. succinate dehydrogenase). We found, that a purified group 1 LEA protein from A. franciscana ( Afr LEA 1.1) helped to retain enzyme activity after desiccation of lactate dehydrogenase (LDH) and dry storage of the enzyme for 1, 3 and 7 days in the presence or absence of bovine serum albumin or trehalose. Increased concentration of purified Afr LEA 1.1, increased the percentage of LDH activity retained after desiccation. To further characterize Afr LEA 1.1, we cloned, expressed, and purified the protein in E. coli . We purified untagged Afr LEA 1.1 protein by affinity chromatography via intein mediated purification with an affinity based chitin‐binding system; a novel protein purification system which utilizes the inducible self‐cleavage activity of protein splicing elements to separate the target protein from the affinity tag. Furthermore, Afr LEA1.1 was expressed in Nicotiana tabacum to investigate if the protein increases drought tolerance of this model plant. Support or Funding Information Supported by NSF IOS‐1456809