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Provedor de dados:  Electron. J. Biotechnol.
País:  Chile
Título:  Group III PLA2 from the scorpion, Mesobuthus tamulus: cloning and recombinant expression in E. coli
Autores:  Hariprasad,Gururao
Saravanan,Kolandaivelu
Singh,Sundararajan Baskar
Das,Utpal
Sharma,Sujata
Kaur,Punit
Singh,Tej Pal
Srinivasan,Alagiri
Data:  2009-07-01
Ano:  2009
Palavras-chave:  Group III phospholipase A2
Mesobuthus tamulus
Recombinant expression
Resumo:  Phospholipases A2 (PLA2) are enzymes that specifically hydrolyze the sn-2 fatty acid acyl bond of phospholipids, producing a free fatty acid and a lyso-phospholipid. We report the cloning and expression of a secretory phospholipase A2 (sPLA2) from Mesobuthus tamulus, Indian red scorpion. The nucleotide sequence codes for a 167 residue enzyme. The open reading frame codes for a 31 amino acid signal peptide followed by a mature portion of the protein. The primary structure shows the calcium binding motif, catalytic residues, 8 highly-conserved cysteines and C-terminal extension which classify it as a group III PLA2. The entire transcript was expressed in Escherichia coli and was purified by metal affinity chromatography under denaturing conditions. The protein was refolded by serial dilutions in the refolding buffer to its active form. Hemolytic assays indicate that the protein adopts a functional conformation. The functional requisites such as optimum pH of 8 and calcium dependency are shown. This report provides a simple but robust methodology for recombinant expression of toxic proteins.
Tipo:  Journal article
Idioma:  Inglês
Identificador:  http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582009000300006
Editor:  Pontificia Universidad Católica de Valparaíso
Formato:  text/html
Fonte:  Electronic Journal of Biotechnology v.12 n.3 2009
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