title: Expression and purification of the membrane enzyme selenoprotein K.
abstract: Selenoprotein K (SelK) is a membrane protein residing in the endoplasmic reticulum. The function of SelK is mostly unknown; however, it has been shown to participate in anti-oxidant defense, calcium regulation and in the endoplasmic reticulum associated protein degradation (ERAD) pathway. In order to study the function of SelK and the role of selenocysteine in catalysis, we have tested heterologous expression of human SelK in E. coli. Consequently, we have developed an over-expression strategy that exploits the maltose binding protein as a fusion partner to stabilize and solubilize SelK. The fusion partner can be cleaved from SelK in the presence of a variety of detergents compatible with structural characterization and the protein purified to homogeneity. SelK acquires a helical secondary structure in detergent micelles, even though it was predicted to be an intrinsically disordered protein due to its high percentage of polar residues. The same strategy was successfully applied to preparation of SelK binding partner - selenoprotein S (SelS). Hence, this heterologous expression and purification strategy can be applied to other members of the membrane enzyme family to which SelK belongs.
authors.affiliation: Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Liu
authors.forename: Prabhavathi
authors.initials: P
authors.lastname: Srinivasan
authors.forename: Diane N
authors.initials: DN
authors.lastname: Pham
authors.forename: Sharon
authors.initials: S
authors.lastname: Rozovsky
mesh_terms: D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology*; D003902:Detergents / Q000737:chemistry; D010450:Endopeptidases / Q000737:chemistry; D004795:Enzyme Stability; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D017279:Selenocysteine / Q000737:chemistry; D051140:Selenoproteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D020539:Sequence Analysis, Protein; D012995:Solubility
chemical_list: D003902:Detergents; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D051140:Selenoproteins; D017279:Selenocysteine; D010450:Endopeptidases; C099488:TEV protease