Antigen binding and cytotoxic properties of a recombinant immunotoxin incorporating the lytic peptide, melittin



Publication Details

Dunn, R., Weston, K., Longhurst, T., Lilley, G., Rivett, D., Hudson, P. and Raison, R. (1996). Antigen binding and cytotoxic properties of a recombinant immunotoxin incorporating the lytic peptide, melittin. Immunotechnology, 2 (3), 229-240.


Background: The majority of immunotoxins studied to date incorporate toxins that act in the cytosol and thus need to be endocytosed by the target cell. An alternative strategy for immunotoxin development is the use of membrane active toxins, such as the pore-forming proteins. Melittin, a 26 amino acid cytolytic peptide from bee venom, is such a protein.

Objectives: We report here the construction, production and functional analysis of a recombinant immunotoxin obtained by fusion of genes which encode an antibody fragment (scFv) with an oligonucleotide encoding melittin.

Study design: The antibody fragment was derived from a murine monoclonal antibody, K121, which recognises a specific epitope (KMA) expressed on the surface of human K myeloma and lymphoma cells, and on human free K Bence Jones protein (BJP). Me1ittin is a 26-amino acid, membrane-lytic peptide which is a major component of bee venom. The scFv of KI21 was constructed by peR to link V H and V L genes via an oligonucleotide which encodes a flexible, hydrophilic peptide. An oligonucleotide encoding melittin and the peptide marker sequence FLAG was fused to the scFv construct using a similar linker peptide. The gene construct (scFv-mel) was inserted into the secretion vector pPOW and expressed in Escherichia coli (TOPP2).

Results: Expression of the recombinant scFv-me1 gene and purification of the protein product was monitored by Western blot analysis. Following purification by anti-FLAG affinity chromatography, the recombinant immunotoxin (scFv-mel) was assessed for antigen binding and for cytotoxic activity by flow cytometry using antigen-expressing and non-expressing cell targets. The scFv-mel was found to exhibit binding and killing properties consistent with the specificity of the original KI21 antibody. Moreover, the cytolytic activity of the scFv-mel was significantly greater on a molar basis than that of native melittin alone.

Conclusion: The data presented here constitute the first report of a melittin-based recombinant immunotoxin and demonstrate that such a membrane active immunotoxin can be synthesised in a bacterial expression.

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