Expression and purification of the transmembrane selenoprotein K

Rozovsky, SharonIn trace amounts, selenium is a necessary element for human development and growth, and selenium aids in the prevention of cancer and decreases the chances of obtaining viruses. By utilizing sulfur pathways, selenium can be incorporated into proteins, as the residue selenocysteine (Sec, U). Sec is similar to its sulfur analog, cysteine (Cys, C). Sec and Cys are similar, but due to Sec’s lower pKa and redox potential, Sec is a far more reactive amino acid and is more likely to be oxidized. There are 25 selenoproteins found in the human genome, and most of selenoproteins play a role in decreasing and/or preventing reactive oxygen species (ROS) from oxidizing the cell. ROS cause oxidative stress, which will lead to damage and eventual death of the cell. One such selenoprotein is Selenoprotein K (Sel K). Sel K is a 94 amino acid protein, where the Sec residue is located at the 92nd position. From previous studies, both the D. melanogaster and human homologs of Sel K protects cells from ROS, and localization studies using green fluorescent protein (GFP) have shown that Sel K spans through the plasma membrane and its C-terminus is in the endoplasmic reticulum (ER). Other than these preliminary studies, Sel K’s structure and function have yet to be determined. In an effort to overexpress Sel K in E. coli, a mutated version of Sel K is used, due to the wild type’s high reactivity from the Sec residue. The Sec residue will be mutated to Cys (U92C), so it will significantly decrease activity. Through a survey of fusion partners that have previously been used to increase protein stability, solubility, and yield, a cytoplasmic Maltose Binding Protein (cMBP) was used in U92C Sel K’s expression and showed the most potential. After amylose purification, TEV proteolytic cleavage occurred between the cMBP and U92C Sel K. Upon further purification, U92C Sel K can be used in future structural and activity studies. Once this system has been optimized and verified, the production of the wild type Sel K will be used for parallel structural and activity studies to further the understanding of this important protein.University of Delaware, Department of Chemistry and BiochemistryM.S