Document Detail


Helix-stabilized Fv (hsFv) antibody fragments: substituting the constant domains of a Fab fragment for a heterodimeric coiled-coil domain.
MedLine Citation:
PMID:  11545598     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Antibody Fv fragments would in principle be useful for a variety of biotechnological applications because of their small size and the possibility to produce them in relatively large amounts in recombinant form; however, their limited stability is a drawback. To solve this problem, both domains are usually fused via a peptide linker to form a single-chain Fv (scFv) fragment, but in some cases this leads to a dimerization. We present an alternative format for stabilizing antibody Fv fragments. The C(H)1 and C(L) domain of the Fab fragment were replaced with a heterodimeric coiled coil (WinZip-A2B1), which had previously been selected using a protein-fragment complementation assay in Escherichia coli. This new antibody format was termed helix-stabilized Fv fragment (hsFv), and was compared to the corresponding Fv, Fab and single-chain Fv format. Bacterial growth and expression of the hsFv was significantly improved compared to the Fab fragment. The hsFv fragment formed a heterodimer of heavy and light chain with the expected molecular mass, also under conditions where the scFv fragment was predominantly dimeric. The hsFv fragment was significantly more stable than the Fv fragment, and nearly as stable as the scFv fragment under the conditions used (80 nM protein concentration). Thus, the format of a helix-stabilized Fv (hsFv) fragment can be a useful alternative to existing recombinant antibody formats, especially in cases where poor expression of Fab fragments or multimerization of scFv fragments is a problem.
Authors:
K M Arndt; K M Müller; A Plückthun
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Publication Detail:
Type:  Comparative Study; Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Journal of molecular biology     Volume:  312     ISSN:  0022-2836     ISO Abbreviation:  J. Mol. Biol.     Publication Date:  2001 Sep 
Date Detail:
Created Date:  2001-09-07     Completed Date:  2001-10-18     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  2985088R     Medline TA:  J Mol Biol     Country:  England    
Other Details:
Languages:  eng     Pagination:  221-8     Citation Subset:  IM    
Copyright Information:
Copyright 2001 Academic Press.
Affiliation:
Biochemisches Institut, Universität Zürich, Winterthurerstr. 190, 8057 Zürich, Switzerland.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Antibodies / chemistry*,  genetics
Dimerization
Escherichia coli / genetics
Immunoglobulin Variable Region / chemistry*,  genetics*,  isolation & purification
Molecular Sequence Data
Mutation
Protein Conformation
Protein Engineering / methods*
Recombinant Proteins / chemistry,  genetics,  metabolism
Chemical
Reg. No./Substance:
0/Antibodies; 0/Immunoglobulin Variable Region; 0/Recombinant Proteins

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