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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
PMID: ISI:A1995RF37600011   
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Abstract
Single-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.
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Times Cited: 31 Article RF376 PROTEIN ENG