This is an archive of papers published by the staff and faculty of Fox Chase Cancer Center. For questions about content, please contact Talbot Research Library
Last updated on
McCartney JE , Tai MS , Hudziak RM , Adams GP , Weiner LM , Jin D , Stafford WF , Liu S , Bookman MA , Laminet AA , Fand I , Houston LL , Oppermann H , Huston JS
Engineering Disulfide-Linked Single-Chain Fv Dimers (Sfv')(2) with Improved Solution and Targeting Properties - Antidigoxin- 26-10 (Sfv')(2) and Anti-C-Erbb-2 741f8 (Sfv')(2) Made by Protein-Folding and Bonded through C-Terminal Cysteinyl Peptides
Protein Engineering. 1995 Mar;8(3) :301-314
AbstractSingle-chain Fv fusions with C-terminal cysteinyl peptides (sFv') have been engineered using model sFv proteins based upon the 26-10 anti-digoxin IgG and 741F8 anti-c-erbB-2 IgG monoclonal antibodies, As part of the 741F8 sFv construction process, the PCR-amplified 741F8 V-H gene was modified in an effort to correct possible primer-induced errors, Genetic replacement of the N-terminal beta-strand sequence of 741F8 V-H With that from the FR1 of anti-c-erbB-2 520C9 V-H resulted in a dramatic improvement of sFv folding yields, Folding in urea- glutathione redox buffers produced active sFv' with a protected C-terminal sulfhydryl, presumably as the mixed disulfide with glutathione, Disulfide-bonded (sFv')(2) homodimers were made by disulfide interchange or oxidation after reductive elimination of the blocking group, Both 26-10 (sFv')(2) and 741F8 (sFv')(2) existed as stable dimers that were well behaved in solution, whereas 741F8 sFv and sFv' exhibited considerable self- association. The 741F8 sFv binds to the extracellular domain (ECD) of the c-erbB-2 oncogene protein, which is often overexpressed in breast cancer and other adenocarcinomas. The recombinant ECD was prepared to facilitate the analysis of 741F8 binding site properties; the cloned ECD gene, modified to encode a C-terminal Ser-Gly-His(6) peptide, was transfected into Chinese hamster ovary cells using a vector that also expressed dihydrofolate reductase to facilitate methotrexate amplification, Optimized cell lines expressed ECD-His(6) at high levels in a cell bioreactor; after isolation by immobilized metal affinity chromatography, final ECD yields were as high as 47 mg/l, An animal tumor model complemented physicochemical studies of 741F8 species and indicated increased tumor localization of the targeted 741F8 (sFv')(2) over other monovalent 741F8 species.
NotesTimes Cited: 31 Article RF376 PROTEIN ENG