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http://hdl.handle.net/1942/3794
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DC Field | Value | Language |
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dc.contributor.author | NICHOLLS, PJ | - |
dc.contributor.author | JOHNSON, VG | - |
dc.contributor.author | Andrew, S.M. | - |
dc.contributor.author | HOOGENBOOM, HR | - |
dc.contributor.author | YOULE, RJ | - |
dc.contributor.author | RAUS, Jef | - |
dc.date.accessioned | 2007-11-29T13:40:03Z | - |
dc.date.available | 2007-11-29T13:40:03Z | - |
dc.date.issued | 1993 | - |
dc.identifier.citation | JOURNAL OF BIOLOGICAL CHEMISTRY, 268(7). p. 5302-5308 | - |
dc.identifier.issn | 0021-9258 | - |
dc.identifier.uri | http://hdl.handle.net/1942/3794 | - |
dc.description.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. | - |
dc.language.iso | en | - |
dc.publisher | AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC | - |
dc.title | CHARACTERIZATION OF SINGLE-CHAIN ANTIBODY (SFV)-TOXIN FUSION PROTEINS PRODUCED INVITRO IN RABBIT RETICULOCYTE LYSATE | - |
dc.type | Journal Contribution | - |
dc.identifier.epage | 5308 | - |
dc.identifier.issue | 7 | - |
dc.identifier.spage | 5302 | - |
dc.identifier.volume | 268 | - |
local.format.pages | 7 | - |
dc.description.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. | - |
local.type.refereed | Refereed | - |
local.type.specified | Article | - |
dc.bibliographicCitation.oldjcat | A1 | - |
dc.identifier.isi | A1993KP88400105 | - |
item.fulltext | No Fulltext | - |
item.fullcitation | NICHOLLS, PJ; JOHNSON, VG; Andrew, S.M.; HOOGENBOOM, HR; YOULE, RJ & RAUS, Jef (1993) CHARACTERIZATION OF SINGLE-CHAIN ANTIBODY (SFV)-TOXIN FUSION PROTEINS PRODUCED INVITRO IN RABBIT RETICULOCYTE LYSATE. In: JOURNAL OF BIOLOGICAL CHEMISTRY, 268(7). p. 5302-5308. | - |
item.contributor | NICHOLLS, PJ | - |
item.contributor | JOHNSON, VG | - |
item.contributor | Andrew, S.M. | - |
item.contributor | HOOGENBOOM, HR | - |
item.contributor | YOULE, RJ | - |
item.contributor | RAUS, Jef | - |
item.accessRights | Closed Access | - |
Appears in Collections: | Research publications |
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