Document Detail


Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli.
MedLine Citation:
PMID:  3045807     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
A biosynthetic antibody binding site, which incorporated the variable domains of anti-digoxin monoclonal antibody 26-10 in a single polypeptide chain (Mr = 26,354), was produced in Escherichia coli by protein engineering. This variable region fragment (Fv) analogue comprised the 26-10 heavy- and light-chain variable regions (VH and VL) connected by a 15-amino acid linker to form a single-chain Fv (sFv). The sFv was designed as a prolyl-VH-(linker)-VL sequence of 248 amino acids. A 744-base-pair DNA sequence corresponding to this sFv protein was derived by using an E. coli codon preference, and the sFv gene was assembled starting from synthetic oligonucleotides. The sFv polypeptide was expressed as a fusion protein in E. coli, using a leader derived from the trp LE sequence. The sFv protein was obtained by acid cleavage of the unique Asp-Pro peptide bond engineered at the junction of leader and sFv in the fusion protein [(leader)-Asp-Pro-VH-(linker)-VL]. After isolation and renaturation, folded sFv displayed specificity for digoxin and related cardiac glycosides similar to that of natural 26-10 Fab fragments. Binding between affinity-purified sFv and digoxin exhibited an association constant [Ka = (3.2 +/- 0.9) x 10(7) M-1] that was about a factor of 6 smaller than that found for 26-10 Fab fragments [Ka = (1.9 +/- 0.2) x 10(8) M-1] under the same buffer conditions, consisting of 0.01 M sodium acetate, pH 5.5/0.25 M urea.
Authors:
J S Huston; D Levinson; M Mudgett-Hunter; M S Tai; J Novotný; M N Margolies; R J Ridge; R E Bruccoleri; E Haber; R Crea
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Proceedings of the National Academy of Sciences of the United States of America     Volume:  85     ISSN:  0027-8424     ISO Abbreviation:  Proc. Natl. Acad. Sci. U.S.A.     Publication Date:  1988 Aug 
Date Detail:
Created Date:  1988-10-05     Completed Date:  1988-10-05     Revised Date:  2009-11-18    
Medline Journal Info:
Nlm Unique ID:  7505876     Medline TA:  Proc Natl Acad Sci U S A     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  5879-83     Citation Subset:  IM    
Affiliation:
Creative Biomolecules, Hopkinton, MA 01748.
Data Bank Information
Bank Name/Acc. No.:
GENBANK/J03850
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MeSH Terms
Descriptor/Qualifier:
Base Sequence
Binding Sites, Antibody*
DNA / analysis
Digoxin / immunology*
Escherichia coli / genetics
Genes, Synthetic*
Immunoglobulin Variable Region / biosynthesis,  genetics*
Molecular Sequence Data
Grant Support
ID/Acronym/Agency:
CA 39870/CA/NCI NIH HHS; HL 19259/HL/NHLBI NIH HHS
Chemical
Reg. No./Substance:
0/Binding Sites, Antibody; 0/Immunoglobulin Variable Region; 20830-75-5/Digoxin; 9007-49-2/DNA
Comments/Corrections

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