title: High-level Expression and Characterization of the Fusion Protein Consisting of Diphtheria Toxin and Human Interleukin 6.
abstract: Diphtheria toxin is a single chain exotoxin of 535 amino acids, secreted from beta Corynebacteriophage diphtheriae. Eukaryotic cells, especially tumor cells are very sensitive to DT. Just one or two molecules of DT can kill a cell. On the surface of some tumor cells, such as human myeloma, heptoma, etc, IL-6 receptor has been demonstrated to be expressed at a very-high level. Selective cytotoxicity mediated by IL-6 receptor could be useful in the targeting therapy of these tumors. Based on this strategy, a hybrid protein consisting of DT and IL-6 was constructed, expressed and characterized. IL-6 cDNA was first modified for constructing the fusion protein of DT/IL-6 and the receptor binding domain of DTDNA was replaced by IL-6 cDNA to obtain the expression vector pdeltaDT/TL-6. After induction by IPTG, the fusion protein was expressed successfully, accounting for 20% of the total bacterial protein. The results of SDS-PAGE and Western blot showed that there was a band of about 64 kD. After preliminary purification, the IL-6 receptor competitive binding test and the cytotoxic activity assay of the deltaDT/TL-6 showed that the fusion protein possessed significant cytotoxic activity to U266 cells; while the cells expressing IL-6 receptors at a medium-level was resistant it to a certain degree.
authors.affiliation: Institute of Virology, the Chinese Academy of Preventive Medicine, Beijing 100052, China.
authors.forename: Xin-Jian
authors.initials: XJ
authors.lastname: Zhang
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.forename: Bao-Yun
authors.initials: BY
authors.lastname: Zhang
authors.forename: Tao
authors.initials: T
authors.lastname: Hong