Abstract
The functional antigen binding region of antidinitrophenol mouse IgA myeloma MOPC 315 has been produced as a single-chain Fv (sFv) protein inE. 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 forK a,app of 1.5 to 2.2×106 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.
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McCartney, J.E., Lederman, L., Drier, E.A. et al. Biosynthetic antibody binding sites: Development of a single-chain Fv model based on antidinitrophenol IgA myeloma MOPC 315. J Protein Chem 10, 669–683 (1991). https://doi.org/10.1007/BF01025718
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DOI: https://doi.org/10.1007/BF01025718