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|Title: ||CHARACTERIZATION OF SINGLE-CHAIN ANTIBODY (SFV)-TOXIN FUSION PROTEINS PRODUCED INVITRO IN RABBIT RETICULOCYTE LYSATE|
|Authors: ||NICHOLLS, PJ|
|Issue Date: ||1993|
|Publisher: ||AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC|
|Citation: ||JOURNAL OF BIOLOGICAL CHEMISTRY, 268(7). p. 5302-5308|
|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.|
|Notes: ||US FDA,CBER,DIV BACTERIAL PROD,BACTERIAL TOXINS LAB,BETHESDA,MD 20892. NCI,EXPTL IMMUNOL BRANCH,BETHESDA,MD 20892. LIMBURGS UNIV CENTRUM,DR L WILLEMS INST,B-3610 DIEPENBEEK,BELGIUM. LIMBURGS UNIV CENTRUM,DEPT WNIF,B-3610 DIEPENBEEK,BELGIUM.NICHOLLS, PJ, NINCDS,SURG NEUROL BRANCH,BIOCHEM SECT,BETHESDA,MD 20892.|
|ISI #: ||A1993KP88400105|
|Type: ||Journal Contribution|
|Appears in Collections: ||Research publications|
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