Renaturation of cobra venom phospholipase A2 expressed from a synthetic gene in Escherichia coli.
Cobra venom (Naja naja naja) phospholipase A2 (PLA2) contains 14 cysteines in the form of 7 disulfide bonds amongst its 119 amino acids. A gene encoding the PLA2 was synthesized and inserted into a bacterial expression vector containing the phage lambda pL promoter. In order to obtain protein without the initiating methionine at the N-terminus, a Factor Xa site was engineered upstream from the PLA2 gene. Upon heat-induction of the cells transformed with the expression plasmid, the protein is produced as insoluble inclusion bodies. The enzyme was partially purified by washing the inclusion bodies with Triton X-100 and urea. The expressed protein was first denatured with 8 M guanidine-HCl and 10 mM DTT. After digestion with Factor Xa, formation of disulfide bonds and refolding into the fully active form was carried out in the presence of cysteine and Ca2+. The renatured recombinant protein was purified by Affi-gel blue column chromatography. The purified recombinant enzyme had the same specific activity as the native enzyme when assayed on a variety of substrates and cross-reacted with antisera prepared against the native enzyme. This is the first report of the expression of a recombinant PLA2 from any venom.
Kelley
M J
MJ
Department of Chemistry, University of California at San Diego, La Jolla 92093-0601.
Crowl
R M
RM
Dennis
E A
EA
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004220:Disulfides / Q000378:metabolism; D004546:Elapid Venoms / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000235:genetics* / Q000378:metabolism; D054467:Phospholipases A2; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D004220:Disulfides; D004546:Elapid Venoms; D011994:Recombinant Proteins; D010741:Phospholipases A; D054467:Phospholipases A2