Construction of a recombinant immunotoxin

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In recent years a number of therapeutically useful immunotoxins have been produced using recombinant gene technology. In general, this involves fusion of a toxin gene with sequence encoding a variety of clinically relevant proteins or peptides. Using these techniques a recombinant immunotoxin has been engineered by fusing the genes encoding an antibody fragment with the sequence of a small cytolytic peptide, melittin. The antibody fragment consists of the antigen binding site derived from a murine monoclonal antibody K- 1-21, which binds to human free kappa light chains and recognises a specific epitope (KMA) expressed on the surface of human myeloma and lymphoma cells. The toxic portion of the molecule is melittin, a 26 amino acid, membrane lytic peptide which is a major component of bee venom. Using PCR a single chain Fv (scFv) was constructed by linking VH and VL genes with an oligonucleotide encoding a flexible, hydrophilic peptide. The melittin gene was synthesised as an oligonucleotide and extended by PCR. Nucleotide sequence encoding a linker peptide was added to the 5' end and a primer encoding a FLAG peptide was used to extend the 3' end. This gene construct was then ligated into the recombinant expression vector, pPOW scFv, to create the fusion gene encoding the recombinant immunotoxin. The gene construct was expressed in the periplasm of E.coli (TOPP2) using the secretion signal pelB . Expression of the foreign protein was monitored by western blot using a monoclonal antibody which recognises the FLAG peptide encoded at the carboxy terminal region of the gene construct. Expression of the recombinant immunotoxin was optimised and the resulting protein was purified using anti-FLAG M2 affinity chromatography. Antigen binding activity was assessed by ELISA and flow cytometry using a human myeloma cell line, HMy2, which expresses the KMA antigen.Binding of the immunotoxin to a control human cell line, K562, which does not express KMA on the cell surface was also assessed. The results indicated that the recombinant immunotoxin retained antigen binding specificity and it was cytotoxic towards the target cell line (HMy2).
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