title: An efficient protocol for the production of tRNA-free recombinant Selenocysteine Synthase (SELA) from Escherichia coli and its biophysical characterization.
abstract: Selenocysteine Synthase (SELA, E.C. 2.9.1.1) from Escherichia coli is a homodecamer pyridoxal-5'-phosphate containing enzyme responsible for the conversion of seryl-tRNA(sec) into selenocysteyl-tRNA(sec) in the biosynthesis of the 21th amino acid, selenocysteine (Sec or U). This paper describes the cloning of the E. coli selA gene into a modified pET29a(+) vector and its expression in E. coli strain WL81460, a crucial modification allowing SELA expression without bound endogenous tRNA(sec). This expression strategy enabled the purification and additional biochemical and biophysical characterization of the SELA decamer. The homogeneous SELA protein was obtained using three chromatographic steps. Size Exclusion Chromatography and Native Gel Electrophoresis showed that SELA maintains a decameric state with molecular mass of approximately 500 kDa with an isoelectric point of 6,03. A predominance of α-helix structures was detected by circular dichroism with thermal stability up to 45 °C. The oligomeric assemblage of SELA was investigated by glutaraldehyde crosslinking experiments indicate that SELA homodecameric structure is the result of a stepwise addition of intermediate oligomeric states and not a direct monomer to homodecamer transition. Our results have contributed to the establishment of a robust expression model for the enzyme free of bound RNA and are of general interest to be taken into consideration in all cases of heterologous/homologous expressions of RNA-binding proteins avoiding the carryover of endogenous RNAs, which may interfere with further biochemical characterizations.
authors.affiliation: Instituto de Física de São Carlos, Universidade de São Paulo, Avenida Trabalhador Saocarlense 400, Zip Code 13560-970, São Carlos, SP, Brazil.
authors.forename: Livia Regina
authors.initials: LR
authors.lastname: Manzine
authors.forename: Alexandre
authors.initials: A
authors.lastname: Cassago
authors.forename: Marco Túlio Alves
authors.initials: MT
authors.lastname: da Silva
authors.forename: Otavio Henrique
authors.initials: OH
authors.lastname: Thiemann
mesh_terms: D001703:Biophysics; D004926:Escherichia coli / Q000201:enzymology*; D008970:Molecular Weight; D017433:Protein Structure, Secondary; D011732:Pyridoxal Phosphate / Q000737:chemistry; D012345:RNA, Transfer, Amino Acid-Specific / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D017279:Selenocysteine / Q000096:biosynthesis / Q000737:chemistry; D014166:Transferases / Q000737:chemistry* / Q000302:isolation & purification*
chemical_list: D012345:RNA, Transfer, Amino Acid-Specific; D011994:Recombinant Proteins; C036928:tRNA, selenocysteine-; D017279:Selenocysteine; D011732:Pyridoxal Phosphate; D014166:Transferases; C060600:selenium transferase