Bioinformatics and Biotechnology

Electronic Journal of Biotechnology ISSN: 0717-3458 Vol. 12 No. 3, Issue of July 15, 2009
© 2009 by Pontificia Universidad Católica de Valparaíso -- Chile Received October 7, 2008 / Accepted March 25, 2009
DOI: 10.2225/vol12-issue3-fulltext-6
RESEARCH ARTICLE

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

Gururao Hariprasad
Department of Biophysics
All India Institute of Medical Sciences
Ansari Nagar, New Delhi - 110 029
India 

Kolandaivelu Saravanan
Department of Biophysics
All India Institute of Medical Sciences
Ansari Nagar, New Delhi - 110 029
India 

Sundararajan Baskar Singh
Department of Biophysics
All India Institute of Medical Sciences
Ansari Nagar, New Delhi - 110 029
India 

Utpal Das
Department of Biophysics
All India Institute of Medical Sciences
Ansari Nagar, New Delhi - 110 029
India 

Sujata Sharma
Department of Biophysics
All India Institute of Medical Sciences
Ansari Nagar, New Delhi - 110 029
India 

Punit Kaur
Department of Biophysics
All India Institute of Medical Sciences
Ansari Nagar, New Delhi - 110 029
India 

Tej Pal Singh
Department of Biophysics
All India Institute of Medical Sciences
Ansari Nagar, New Delhi - 110 029
India

Alagiri Srinivasan*
Department of Biophysics
All India Institute of Medical Sciences
Ansari Nagar, New Delhi - 110 029
India
Tel: 91 11 2659 4240
Fax: 91 11 2658 8663
E-mail: srini@aiims.ac.in

*Corresponding author

Financial support: Department of Science and Technology as a grant to GH (No. SR/FT/L-18/2005).

Keywords: group III phospholipase A2, Mesobuthus tamulus, recombinant expression.

Abbreviations:

Ni-NTA: nickel nitrilo tri aceticacid
PBS: phosphate buffered saline
PLA2: phospholipase A2
PMSF: phenyl methane sulphonyl fluoride
RBC: red blood cells
SDS: sodium dodecyl sulphate
sPLA2: secretory phospholipase A2

Abstract   Full Text

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.

Supported by UNESCO / MIRCEN network