Expression of fully active ammodytoxin A, a potent presynaptically neurotoxic phospholipase A2, in Escherichia coli

FEBS Letters

Pungerčar, Jože¹  Gubenšek, Franc¹  Liang, Ning-Sheng¹  Štrukelj, Borut¹  Križaj, Igor¹ 

[1]Department of Biochemistry and Molecular Biology, Jožef Stefan Institute, Jamova 39, PO Box 100, 61111 Ljubljana, Slovenia

关键词: Phospholipase A2;    Neurotoxin;    Refolding;    Expression;    Snake;    Vipera ammodytes;    cDNA;    complementary DNA;    DTT;    1;    4-dithiothreitol;    EDTA;    disodium ethylenediaminotetraacetate;    GdnHCl;    guanidine hydrochloride;    IPTG;    isopropyl-β-d-thiogalactoside;    kDa;    kilodalton;    NTSB;    disodium 2-nitro-5-thiosulfobenzoate;    PAGE;    polyacrylamide gel electrophoresis;    PLA2;    phospholipase A2;    SDS;    sodium dodecyl sulfate;    TFA;    trifluoroacetic acid;   

DOI  :  10.1016/0014-5793(93)81679-T

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

A cDNA encoding the most presynaptically neurotoxic phospholipase A₂, ammodytoxin A, from the venom of the long-nosed viper (Vipera ammodytes ammodytes) has been expressed in Escherichia coli. Ammodytoxin A was produced as a fusion protein with the 81 N-terminal residues of adenylate kinase followed by the tetrapeptide recognition site for factor Xa (IEGR) just preceding the first amino acid residue of the toxin. The fusion protein was expressed under the control of tac promoter without IPTG induction in the form of insoluble inclusion bodies. It was dissolved in guanidine hydrochloride, S-sulfonated and refolded in a reoxidation mixture including a reduced/oxidized glutathione redox couple. Ammodytoxin A was fully activated by limited hydrolysis with trypsin that preferentially cleaves the fusion protein at the factor Xa recognition site and purified by cation-exchange chromatography. The correct N-terminus was confirmed by protein sequencing. Recombinant ammodytoxin A has been proved to be indistinguishable from the native toxin in its enzymatic activity and toxicity.

【 授权许可】

Unknown