Characterization of single-chain antibody (sFv)-toxin fusion proteins produced in vitro in rabbit reticulocyte lysate.

Nicholls, P. J. and Johnson, V. G. and Hoogenboom, H. R. and Raus, J. C. M. and Youle, R. J. (1993) Characterization of single-chain antibody (sFv)-toxin fusion proteins produced in vitro in rabbit reticulocyte lysate. Journal of Biological Chemistry, 268 (7). pp. 5302-5308. ISSN 1083-351X

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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.

Item Type:
Journal Article
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Journal of Biological Chemistry
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?? biochemistrycell biologymolecular biologyge environmental sciences ??
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20 Jan 2009 15:24
Last Modified:
15 Jul 2024 10:11