Open AccessProtein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli.
Author(s) -
James S. Huston,
Dennis J. Levinson,
Meredith Mudgett-Hunter,
Mei-Sheng Tai,
James F. Novotny,
Michael N. Margolies,
Richard J. Ridge,
Robert E. Bruccoleri,
Edgar Haber,
Roberto Crea
Publication year - 1988
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.85.16.5879
Subject(s) - fusion protein , escherichia coli , linker , chemistry , immunoglobulin light chain , microbiology and biotechnology , peptide sequence , amino acid , biochemistry , peptide , biology , antibody , recombinant dna , gene , computer science , immunology , operating system
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.