An efficient protocol for the production of tRNA-free recombinant Selenocysteine Synthase (SELA) from Escherichia coli and its biophysical characterization

[Display omitted] ► Recombinant SELA present a bound RNA compromising functional analysis. ► A simple expression system was developed in a tRNAsec free Escherichia coli strain. ► SELA molecular characterization shows it is equivalent to the native enzyme. ► Crosslinking experiments show that homodec...

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Published in:Protein expression and purification 2013-03, Vol.88 (1), p.80-84
Main Authors: Manzine, Livia Regina, Cassago, Alexandre, da Silva, Marco Túlio Alves, Thiemann, Otavio Henrique
Format: Article
Language:English
Subjects:
Biophysics
Circular dichroism
Escherichia coli - enzymology
Fluorescence
Molecular Weight
Protein Structure, Secondary
Pyridoxal Phosphate - chemistry
Pyridoxal-5′-phosphate
Recombinant Proteins - biosynthesis
Recombinant Proteins - chemistry
Recombinant Proteins - isolation & purification
RNA, Transfer, Amino Acid-Specific - chemistry
Selenocysteine - biosynthesis
Selenocysteine - chemistry
Selenocysteine Synthase
Transferases - chemistry
Transferases - isolation & purification
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Summary:[Display omitted] ► Recombinant SELA present a bound RNA compromising functional analysis. ► A simple expression system was developed in a tRNAsec free Escherichia coli strain. ► SELA molecular characterization shows it is equivalent to the native enzyme. ► Crosslinking experiments show that homodecamer formation is a sequential pathway. 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-tRNAsec into selenocysteyl-tRNAsec 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 500kDa 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.
ISSN:1046-5928
1096-0279
DOI:10.1016/j.pep.2012.12.005