Group III PLA2 from the scorpion, Mesobuthus tamulus: cloning and recombinant expression in E. coli

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 nucleot...

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Autores principales:	Hariprasad,Gururao, Saravanan,Kolandaivelu, Singh,Sundararajan Baskar, Das,Utpal, Sharma,Sujata, Kaur,Punit, Singh,Tej Pal, Srinivasan,Alagiri
Lenguaje:	English
Publicado:	Pontificia Universidad Católica de Valparaíso 2009
Materias:	group III phospholipase A2 Mesobuthus tamulus recombinant expression
Acceso en línea:	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582009000300006
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spelling	oai:scielo:S0717-345820090003000062010-04-01Group III PLA2 from the scorpion, Mesobuthus tamulus: cloning and recombinant expression in E. coliHariprasad,GururaoSaravanan,KolandaiveluSingh,Sundararajan BaskarDas,UtpalSharma,SujataKaur,PunitSingh,Tej PalSrinivasan,Alagiri group III phospholipase A2 Mesobuthus tamulus recombinant expression 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.info:eu-repo/semantics/openAccessPontificia Universidad Católica de ValparaísoElectronic Journal of Biotechnology v.12 n.3 20092009-07-01text/htmlhttp://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582009000300006en10.4067/S0717-34582009000300006
institution	Scielo Chile
collection	Scielo Chile
language	English
topic	group III phospholipase A2 Mesobuthus tamulus recombinant expression
spellingShingle	group III phospholipase A2 Mesobuthus tamulus recombinant expression Hariprasad,Gururao Saravanan,Kolandaivelu Singh,Sundararajan Baskar Das,Utpal Sharma,Sujata Kaur,Punit Singh,Tej Pal Srinivasan,Alagiri Group III PLA2 from the scorpion, Mesobuthus tamulus: cloning and recombinant expression in E. coli
description	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.
author	Hariprasad,Gururao Saravanan,Kolandaivelu Singh,Sundararajan Baskar Das,Utpal Sharma,Sujata Kaur,Punit Singh,Tej Pal Srinivasan,Alagiri
author_facet	Hariprasad,Gururao Saravanan,Kolandaivelu Singh,Sundararajan Baskar Das,Utpal Sharma,Sujata Kaur,Punit Singh,Tej Pal Srinivasan,Alagiri
author_sort	Hariprasad,Gururao
title	Group III PLA2 from the scorpion, Mesobuthus tamulus: cloning and recombinant expression in E. coli
title_short	Group III PLA2 from the scorpion, Mesobuthus tamulus: cloning and recombinant expression in E. coli
title_full	Group III PLA2 from the scorpion, Mesobuthus tamulus: cloning and recombinant expression in E. coli
title_fullStr	Group III PLA2 from the scorpion, Mesobuthus tamulus: cloning and recombinant expression in E. coli
title_full_unstemmed	Group III PLA2 from the scorpion, Mesobuthus tamulus: cloning and recombinant expression in E. coli
title_sort	group iii pla2 from the scorpion, mesobuthus tamulus: cloning and recombinant expression in e. coli
publisher	Pontificia Universidad Católica de Valparaíso
publishDate	2009
url	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582009000300006
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Group III PLA2 from the scorpion, Mesobuthus tamulus: cloning and recombinant expression in E. coli

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