Refolding and purification of Bothropstoxin-I, a Lys49-phospholipase A2 homologue, expressed as inclusion bodies in Escherichia coli.
Hydrolysis of phospholipids by Group II phospholipase A2 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(2) 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.
Ward
R J
RJ
Department of Chemistry, Protein Chemistry Center, Universidiade de São Paulo, São Paulo, Brazil. rjward@fmrp.usp.br
de Oliveira
A H
AH
Bortoleto
R K
RK
Rosa
J C
JC
Faça
V M
VM
Greene
L J
LJ
D019943:Amino Acid Substitution; D000818:Animals; D017837:Bothrops; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular / Q000379:methods; D003432:Cross-Linking Reagents; D003435:Crotalid Venoms / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine; D002479:Inclusion Bodies / Q000378:metabolism; D008081:Liposomes; D008239:Lysine; D010741:Phospholipases A / Q000737:chemistry*; D054467:Phospholipases A2; D017510:Protein Folding; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013050:Spectrometry, Fluorescence; D013501:Surface-Active Agents
D003432:Cross-Linking Reagents; D003435:Crotalid Venoms; D018076:DNA, Complementary; D008081:Liposomes; D013501:Surface-Active Agents; C078097:bothropstoxin; D006639:Histidine; D010741:Phospholipases A; D054467:Phospholipases A2; D008239:Lysine