Construction, expression, purification and functional analysis of recombinant 6C6 immunotoxin to human breast-tumor...

The 28 ku membrane protein is usually over-expressed in human breast cancer and other tumor cells. It could be a target for tumor therapy. By using genetic engineering techniques, a 6C6 immunotoxin (ScFv6C6-PE40) was constructed by joining the 6C6 single-chain antibody (ScFv6C6) with the truncatedPseudomonas exotoxin A (PE40). ScFv6C6 contains both the heavy- and light-chain variable domains of 6C6 monoclonal antibody, which specifically recognizes the 28 ku protein. The bacterial expression level of 6C6 immunotoxin is 3.3%, about 5.5 mg/mL bacterial lysate. Using single-step HisTrap (Ni(2+) chelating) column chromatography, the recombinant peptide was obtained with a purity of 33.2%. This bacterial expressed 6C6 immunotoxin binds to MDA-231 human breast-tumor cell surface and kills these cells with a median lethal dosage of 92 ng/mL.