Properties of chimeric toxins with two recognition domains: interleukin 6 and transforming growth factor alpha at different locations in Pseudomonas exotoxin.
Pseudomonas exotoxin (PE) is a potent cytotoxic agent that is composed of 613 amino acids arranged into three major domains. We have previously identified two positions where ligands can successfully be placed in PE to direct it to cells with specific surface receptors. One site is at the amino terminus and the other is close to but not at the C-terminus. To examine the possibility of constructing oncotoxins with two different recognition elements that will bind to two different receptors, we have placed cDNAs encoding either transforming growth factor alpha (TGF alpha) or interleukin 6 (IL6) at the 5' end of a PE gene and also inserted a cDNA encoding TGF alpha near the 3' end of the PE gene. The plasmids encoding these chimeric toxins were expressed in Escherichia coli and the chimeric proteins purified to near homogeneity. In all the new toxins, the TGF alpha near the C-terminus was inserted after amino acid 607 of PE and followed by amino acids 604-613 so that the correct PE C-terminus (REDLK) was preserved. For each chimera, the toxin portion was either PE4E, in which the cell binding domain (domain Ia) is mutated, PE40, in which domain Ia is deleted, or PE38, in which domain Ia and part of domain Ib are deleted. These derivatives of PE do not bind to the PE receptor and allow 607, 355, or 339 amino acids, respectively, between the two ligands.(ABSTRACT TRUNCATED AT 250 WORDS)
Kreitman
R J
RJ
Laboratory of Molecular Biology, DCBDC, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Siegall
C B
CB
Chaudhary
V K
VK
FitzGerald
D J
DJ
Pastan
I
I
D036002:ADP Ribose Transferases; D000818:Animals; D001665:Binding Sites; D066246:ErbB Receptors / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005098:Exotoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D000922:Immunotoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D015850:Interleukin-6 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D007928:Lethal Dose 50; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins; D016211:Transforming Growth Factor alpha / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D014407:Tumor Cells, Cultured
D005098:Exotoxins; C064195:IL-6-PE40 protein, chimeric; D000922:Immunotoxins; D015850:Interleukin-6; D011993:Recombinant Fusion Proteins; D016211:Transforming Growth Factor alpha; C062005:transforming growth factor type alpha-Pseudomonas exotoxin A; D036002:ADP Ribose Transferases; D066246:ErbB Receptors