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.
Full text not available from this repository.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.
| Item Type: | Article |
|---|---|
| Journal or Publication Title: | Journal of Biological Chemistry |
| Subjects: | G Geography. Anthropology. Recreation > GE Environmental Sciences |
| Departments: | UNSPECIFIED |
| ID Code: | 22661 |
| Deposited By: | ep_ss_importer |
| Deposited On: | 20 Jan 2009 15:24 |
| Refereed?: | No |
| Published?: | Published |
| Last Modified: | 26 Jul 2012 16:04 |
| Identification Number: | |
| URI: | http://eprints.lancs.ac.uk/id/eprint/22661 |
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