dc.creatorManzine, Livia Regina
dc.creatorCassago, Alexandre
dc.creatorSilva, Marco Túlio Alves da
dc.creatorThiemann, Otavio Henrique
dc.date.accessioned2014-05-22T23:22:01Z
dc.date.accessioned2018-07-04T16:45:59Z
dc.date.available2014-05-22T23:22:01Z
dc.date.available2018-07-04T16:45:59Z
dc.date.created2014-05-22T23:22:01Z
dc.date.issued2013-03
dc.identifierProtein Expression and Purification, Amsterdam : Elsevier, v. 88, n. 1, p. 80-84, Mar. 2013
dc.identifier1046-5928
dc.identifierhttp://www.producao.usp.br/handle/BDPI/44994
dc.identifier10.1016/j.pep.2012.12.005
dc.identifier.urihttp://repositorioslatinoamericanos.uchile.cl/handle/2250/1640072
dc.description.abstractSelenocysteine 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 tRNAsec. 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.
dc.languageeng
dc.publisherElsevier
dc.publisherAmsterdam
dc.relationProtein Expression and Purification
dc.rightsCopyright Elsevier Inc.
dc.rightsrestrictedAccess
dc.subjectSelenocysteine synthase
dc.subjectPyridoxal-5′-phosphate
dc.subjectCircular dichroism
dc.subjectFluorescence
dc.titleAn efficient protocol for the production of tRNA-free recombinant Selenocysteine Synthase (SELA) from Escherichia coli and its biophysical characterization
dc.typeArtículos de revistas


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