Document Detail


Selecting highly structure-specific antibodies using structured synthetic mimics of the cystine knot protein sclerostin.
MedLine Citation:
PMID:  22454505     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
Antibodies directed against specific regions of a protein have traditionally been raised against full proteins, protein domains or simple unstructured peptides, containing contiguous stretches of primary sequence. We have used a new approach of selecting antibodies against restrained peptides mimicking defined epitopes of the bone modulator protein sclerostin, which has been identified as a negative regulator of the Wnt pathway. For a fast exploration of activity defining epitopes, we produced a set of synthetic peptide constructs mimicking native sclerostin, in which intervening loops from the cystine-knot protein sclerostin were truncated and whose sequences were optimized for fast and productive refolding. We found that the second loop within the cystine knot could be replaced by unnatural sequences, both speeding up folding, and increasing yield. Subsequently, we used these constructs to pan the HuCAL phage display library for antibodies capable of binding the native protein, thereby restricting recognition to the desired epitope regions. It is shown that the antibodies that were obtained recognize a complex epitope in the protein that cannot be mimicked with linear peptides. Antibodies selected against peptides show similar recognition specificity and potency as compared with antibodies obtained from full-length recombinant protein.
Authors:
J W Back; C Frisch; K Van Pee; V Boschert; R van Vught; W Puijk; T D Mueller; A Knappik; P Timmerman
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-3-27
Journal Detail:
Title:  Protein engineering, design & selection : PEDS     Volume:  -     ISSN:  1741-0134     ISO Abbreviation:  -     Publication Date:  2012 Mar 
Date Detail:
Created Date:  2012-3-28     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  101186484     Medline TA:  Protein Eng Des Sel     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Affiliation:
Pepscan Therapeutics, Zuidersluisweg 2, 8203RC Lelystad, The Netherlands.
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