CLONING AND EXPRESSION OF A POTENTIAL PRESYNAPTIC NEUROTOXIN PHOSPHOLIPASE A2-TYPE FROM THE CORAL SNAKE Micrurus corallinus

Mini-Symposium - Abstracts

15 CLONING AND EXPRESSION OF A POTENTIAL PRESYNAPTIC NEUROTOXIN PHOSPHOLIPASE A2-TYPE FROM THE CORAL SNAKE Micrurus corallinus

A. ASSNI , E. CARMONA , A.C.M.R. CAMPOS , I. RAW, P.L. HO

Center of Biotechnology , Laboratory of Pharmacology and Immunochemistry , Butantan Institute, 05503-900, São Paulo, SP, Brazil.

We have recently described the isolation of several cDNAs coding for potential toxins from the venom gland of Micrurus corallinus, a South American coral snake (Ho et al., J. Toxicol Toxin Reviews 14; 327, 1995). One of these cDNAs (clone V2) shares high structural homology with neurotoxic PLA2-like proteins, such as those characterized from Arpysurus laeyis and Notechis scutatus scutatus. The deduced protein from clone V2 cDNA has shown that it should belong to monomeric class I PLA2, and is predicted to be a Ca dependent enzyme. The deduced protein also possesses potential N-glycosilation and phosphorylation sites. In order to uncover the biological function of this protein, we first tried to express this cDNA using the methylotropic yeast Pichia pastoris (Invitrogen, USA). However, our attempts were unsuccessful. Then, we changed the expression system to an E. coli based vector. The cDNA from clone V2 was amplified by PCR in order to remove the signal peptide and also to include some restriction sites at 5' end to directionally clone into the pRSET C vector (Invitrogen, USA). The pRSET C vector uses the T7 RNA polymerase promoter to drive the expression of the recombinant proteins. The resulted construction pRSET C-PLA2 was used to transform E. coli BL21(DE3) that has integrated in its genome the T7 RNA polymerase gene under the control of the lac UV5 promoter. Thus, the transformants were induced with 0.4 mM IPTG and the expression was assessed by SDS-PAGE analysis. The positive clones were selected. The recombinant protein was expressed as inclusion bodies that could be solubilyzed by 8M urea. In this condition, the expressed PLA2 was partially purified by Ni+2-charged Sepharose. Further characterization of the recombinant PLA2 is now under way.

Financial support: FAPESP, CNPq and Fundação Butantan.

CORRESPONDENCE TO:

Dr. Paulo Lee Ho - Instituto Butantan, Centro de Biotecnologia, Avenida Vital Brazil, 1500, CEP 05503-900, São Paulo, SP, Brasil. email: [e-mail]

Publication Dates

• Publication in this collection
  08 Jan 1999
• Date of issue
  1997