Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli
| Autor: | M S Tai, Jirí Novotný, D. Levinson, James S. Huston, R Crea, R J Ridge, Robert E. Bruccoleri, Edgar Haber, Michael N. Margolies, Meredith Mudgett-Hunter |
|---|---|
| Rok vydání: | 1988 |
| Předmět: |
chemistry.chemical_classification
Digoxin Multidisciplinary Base Sequence Chemistry Oligonucleotide viruses Molecular Sequence Data Immunoglobulin Variable Region DNA Protein engineering biochemical phenomena metabolism and nutrition medicine.disease_cause Molecular biology Fusion protein Amino acid Escherichia coli Genes Synthetic medicine Peptide bond Binding Sites Antibody Binding site Linker Research Article |
| Zdroj: | Proceedings of the National Academy of Sciences. 85:5879-5883 |
| ISSN: | 1091-6490 0027-8424 |
| DOI: | 10.1073/pnas.85.16.5879 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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