Purification and characterization of IL6-PE4E, a recombinant fusion of interleukin 6 with Pseudomonas exotoxin.
We have developed a procedure to purify the recombinant fusion toxin IL6-PE4E from Escherichia coli which results in a high yield of fully active monomeric protein of high purity and very low endotoxin content. The chimeric toxin is composed of human interleukin 6 (IL6) fused to a derivative of Pseudomonas exotoxin (PE) containing mutations in the binding domain which prevent binding to the PE receptor. In a typical preparation, 20 g of E. coli cells expressing the plasmid encoding IL6-PE4E were treated with lysozyme and washed repeatedly with detergent (Triton X-100), to obtain 500 mg of inclusion bodies. The recombinant protein was denatured and reduced in guanidine hydrochloride solution containing dithioerythritol and refolded in a redox buffer containing oxidized glutathione and L-arginine. After purification of the dialyzed protein by anion-exchange, polymyxin B, and sizing chromatography, we obtained 100 mg (20% of recombinant protein) of purified monomer with 0.6-2.5 endotoxin units/mg of protein. Amino terminal sequencing confirmed the first 20 amino acids. IL6-PE4E purified in this manner was fully cytotoxic toward human multiple myeloma, hepatoma, epidermoid carcinoma, and prostate carcinoma cell lines. After intravenous injection into mice, we found the dose-limiting toxicity to be to the liver, by measurement of serum transaminases and histologic evaluation of the liver. The LD50 was 450 micrograms/kg. We conclude that IL6-PE4E can be purified efficiently for preclinical testing.
Kreitman
R J
RJ
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Pastan
I
I
D036002:ADP Ribose Transferases; D000818:Animals; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000737:chemistry; D005098:Exotoxins / Q000302:isolation & purification* / Q000633:toxicity*; D005260:Female; D006801:Humans; D015850:Interleukin-6 / Q000302:isolation & purification* / Q000633:toxicity*; D007928:Lethal Dose 50; D051379:Mice; D008819:Mice, Nude; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000633:toxicity*
D005098:Exotoxins; C064195:IL-6-PE40 protein, chimeric; D015850:Interleukin-6; D011993:Recombinant Fusion Proteins; D036002:ADP Ribose Transferases