Biosynthetic antibody binding sites: Development of a single-chain Fv model based on antidinitrophenol IgA myeloma MOPC 315
| Autor: | John E. McCartney, Lynne Lederman, Gay-May Wu, James S. Huston, Roberta S. Batorsky, Hermann Oppermann, Eric A. Drier, Nancy A. Cabral-Denison |
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| Rok vydání: | 1991 |
| Předmět: |
Protein Conformation
Recombinant Fusion Proteins Molecular Sequence Data Biochemistry law.invention Mice Antigen law Animals Bioorganic chemistry Amino Acid Sequence Binding site Base Sequence biology Chemistry DNA Protein engineering Fusion protein Molecular biology Recombinant DNA biology.protein Specific activity Binding Sites Antibody Antibody Multiple Myeloma Dinitrophenols |
| Zdroj: | Journal of Protein Chemistry. 10:669-683 |
| ISSN: | 1573-4943 0277-8033 |
| DOI: | 10.1007/bf01025718 |
| Popis: | The functional antigen binding region of antidinitrophenol mouse IgA myeloma MOPC 315 has been produced as a single-chain Fv (sFv) protein in E. coli. Recombinant 315 proteins included sFv alone, a bifunctional fusion protein with amino-terminal fragment B (FB) of staphylococcal protein A, and a two-chain 315 Fv fragment. Successful refolding of the 315 sFv required formation of disulfide bonds while the polypeptide was in a denatured state, as previously observed for the parent Fv fragment. Affinity-purified recombinant 315 proteins showed full recovery of specific activity, with values for Ka,app of 1.5 to 2.2 x 10(6) M-1, equivalent to the parent 315 Fv fragment. As observed for natural 315 Fv, the sFv region of active FB-sFv315 fusion protein was resistant to pepsin treatment, whereas inactive protein was readily degraded. These experiments will allow the application of protein engineering to the 315 single-chain Fv; such studies can advance structure-function studies of antibody combining sites and lead to an improved understanding of single-chain Fv proteins. |
| Databáze: | OpenAIRE |
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