title: Characterization of single-chain antibody (sFv)-toxin fusion proteins produced in vitro in rabbit reticulocyte lysate.
abstract: Chimeric proteins consisting of a fusion between binding-deficient mutants of diphtheria toxin (DT) or Pseudomonas exotoxin A (PE) and a single-chain antibody (E6 sFv) against the human transferrin receptor (TfnR) were expressed in a rabbit reticulocyte lysate system. Molecules utilizing PE40 (the carboxyl terminus 40 kDa of PE, lacking the binding domain) exhibited significant E6 sFv-mediated, cell type-specific cytotoxicity (IC50 1 x 10(-10) M) against a human erythroleukemia-derived cell line, K562. In contrast, a fusion protein between the same sFv and a DT mutant, DTM1 (containing two amino acid substitutions in the binding domain [S(508)F, S(525)F]) was not significantly cytotoxic, despite being enzymatically active. A tripartite protein in the form NH2-DTM1-E6 sFv-PE40-COOH exhibited cytotoxicity comparable to that of the PE40-sFv fusion (IC50 1 x 10(-10) M), suggesting that the deficit in activity of DTM1-sFv is not a function of misfolding of the sFv moiety or of a reduced ability to bind TfnR. In contrast to DTM1-E6 sFv, a fusion protein between a second DT mutant, CRM 107 [S(525)F], and the E6 sFv was specifically cytotoxic (IC50 1 x 10(-9) M), and toxicity could be blocked by addition of excess E6 antibody. The cell-free in vitro expression system we describe is rapid and may be used to express functional toxin-sFv fusion proteins. No protein refolding procedures are required, and the technique may be used to express proteins which, due to restrictions imposed on manipulation of toxin-encoding genes in Escherichia coli, could not be produced by more conventional methods.
authors.affiliation: Biochemistry Section, National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: P J
authors.initials: PJ
authors.lastname: Nicholls
authors.forename: V G
authors.initials: VG
authors.lastname: Johnson
authors.forename: S M
authors.initials: SM
authors.lastname: Andrew
authors.forename: H R
authors.initials: HR
authors.lastname: Hoogenboom
authors.forename: J C
authors.initials: JC
authors.lastname: Raus
authors.forename: R J
authors.initials: RJ
authors.lastname: Youle
mesh_terms: D036002:ADP Ribose Transferases; D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002460:Cell Line; D002470:Cell Survival; D004247:DNA; D004167:Diphtheria Toxin / Q000235:genetics / Q000378:metabolism*; D005098:Exotoxins / Q000235:genetics / Q000378:metabolism*; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology*; D000922:Immunotoxins / Q000235:genetics / Q000276:immunology* / Q000633:toxicity; D008969:Molecular Sequence Data; D010957:Plasmids; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism*; D014709:Vero Cells; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D004247:DNA; D004167:Diphtheria Toxin; D005098:Exotoxins; D007128:Immunoglobulin Fragments; D000922:Immunotoxins; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A