title: A circularly permuted recombinant interleukin 4 toxin with increased activity.
abstract: Fusion of ligands such as growth factors to other proteins often dramatically reduces the affinity of the ligand for its receptor. With recombinant DNA techniques, the attachment point between the two proteins has until now been restricted to either the amino or the carboxyl terminus of the ligand. However, binding may be greatly compromised if both ends are close to the site at which the ligand binds to its receptor. To construct a single-chain growth factor fusion protein with the connection at a new site on the growth factor, we constructed a DNA fragment encoding circularly permuted interleukin 4 (IL4), termed IL4(38-37). This was accomplished by placing a start codon before position 38, connecting codons 1 and 129 with a sequence encoding a peptide linker, and placing a stop codon after codon 37 of IL4. IL4(38-37) was fused via its new carboxyl terminus, Lys37, to a truncated form of Pseudomonas exotoxin. The purified circularly permuted IL4-toxin bound to the IL4 receptor with 10-fold higher affinity than an IL4-toxin in which the toxin was fused to the carboxyl terminus of IL4. Circular permuteins of growth factors can improve the effectiveness of recombinant fusion proteins, because the junction can be moved to a site on the growth factor which allows it to bind with higher affinity.
authors.affiliation: Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
authors.forename: R J
authors.initials: RJ
authors.lastname: Kreitman
authors.forename: R K
authors.initials: RK
authors.lastname: Puri
authors.forename: I
authors.initials: I
authors.lastname: Pastan
mesh_terms: D036002:ADP Ribose Transferases; D001426:Bacterial Proteins / Q000378:metabolism* / Q000633:toxicity; D001427:Bacterial Toxins; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon; D004247:DNA; D005098:Exotoxins / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D006801:Humans; D015847:Interleukin-4 / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000633:toxicity; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D003062:Codon; D004247:DNA; D005098:Exotoxins; D015847:Interleukin-4; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C059989:IL-4-PE40 protein, recombinant