Refolding and purification of bothropstoxin-1, a Lys49 - Phospholipase A(2) homologue, expressed as inclusion bodies in Escherichia coli

Nenhuma Miniatura disponível

Data

2001-02-01

Autores

Ward, R. J.
de Oliveira, AHC
Bortoleto, R. K.
Rosa, J. C.
Faca, V. M.
Greene, L. J.

Título da Revista

ISSN da Revista

Título de Volume

Editor

Academic Press Inc.

Resumo

Hydrolysis of phospholipids by Group II phospholipase A(2) enzymes involves a nucleophilic attack on the sn-2 ester bond by the His48 residue and stabilization of the reaction intermediate by a Ca2+ ion cofactor bound to the Asp49 residue in the protein active site region, Bothropstoxin-I (BthTX-I) is a PLA, variant present in the venom of the snake Bothrops jararacussu which shows a Asp49 to Lys substitution and which lacks hydrolytic activity yet damages artificial membranes by a noncatalytic Ca2+-independent mechanism. In order to better characterize this unusual mechanism of membrane damage, we have established an expression system for BthTX-I in Escherichia coli. The DNA-coding sequence for BthTX-I was subcloned into the vector pET11-d, and the BthTX-I was expressed as inclusion bodies in E, coli BL21(DE3). The native BthTX-I contains seven disulfide bonds, and a straightforward protocol has been developed to refold the recombinant protein at high protein concentration in the presence of surfactants using a size-exclusion chromatography matrix. After refolding, recovery yields of 2.5% (corresponding to 4-5 mg of refolded recombinant BthTX-I per liter of bacterial culture) were routinely obtained. After refolding, identical fluorescent and circular dichroism spectra were obtained for the recombinant BthTX-I compared to those of the native protein. Furthermore, the native and refolded recombinant protein demonstrated identical membrane-damaging properties as evaluated by measuring the release of an entrapped fluorescent marker from liposomes, (C) 2001 Academic Press.

Descrição

Palavras-chave

Como citar

Protein Expression and Purification. San Diego: Academic Press Inc., v. 21, n. 1, p. 134-140, 2001.