High-level expression in Escherichia coli and rapid purification of enzymatically active honey bee venom phospholipase A 2

Abstract

Bee venom phospholipase A 2 (BV-PLA2) is a hydrolytic enzyme that specifically cleaves the sn-2 acyl bond of phospholipids at the lipid/water interface. The same enzyme is also believed to be responsible for some systemic anaphylactic reactions in bee venom sensitized individuals. To study the structure/function relationships of this enzyme and to define the molecular determinants responsible for its allergenic potential, a synthetic gene encoding the mature form of BV-PLA2 was expressed in Escherichia coli. This enzyme was produced as a fusion protein with a 6xHis-tag on its amino-terminus yielding 40–50 mg of fusion protein per 1 of culture after metal ion affinity chromatography. A kallikrein protease recognition site was engineered between the 6xHis-tag and the amino-terminus of the enzyme allowing isolation of the protein with its correct N-terminus. Recombinant affinity purified BV-PLA2 was refolded, purified to homogeneity, and cleaved with kallikrein, resulting in a final yield of 8–9 mg of active enzyme per 1 of culture. The enzymatic and immunological properties of the recombinant BV-PLA2 are identical to enzyme isolated from bee venom indicating a native-like folding of the protein.