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http://dx.doi.org/10.7845/kjm.2018.8061

The development of anti-DR4 single-chain Fv (ScFv) antibody fused to Streptavidin  

Kim, Seo Woo (Division of Biohealth Science, College of Natural Sciences, Changwon National University)
Wu, Sangwook (Department of Physics, College of Natural Sciences, Pukyong National University)
Kim, Jin-Kyoo (Division of Biohealth Science, College of Natural Sciences, Changwon National University)
Publication Information
Korean Journal of Microbiology / v.54, no.4, 2018 , pp. 330-342 More about this Journal
Abstract
The Streptavidin and Biotin system has been studied most extensively as the high affinity non-covalent binding of Biotin to STR ($K_D=10^{-14}M$) and four Biotin binding sites in tetrameric Streptavidin makes this system useful for the production of multivalent antibody. For the application of this system, we cloned Streptavidin amplified from Streptomyces avidinii chromosome by PCR and fused to gene of hAY4 single-chain Fv antibody specific to death receptor 4 (DR4) which is a receptor for tumor necrosis factor ${\alpha}$ related apoptosis induced ligand. The hAY4 single-chain Fv antibody fused to Streptavidin expressed in Escherichia coli showed 43 kDa monomer in heated SDS-PAGE. However, this fusion protein shown in both non-heated SDS-PAGE and Size-exclusion chromatography exhibited 172 kDa as a tetramer suggesting that natural tetramerization of Streptavidin by non-covalent association induced hAY4 single-chain Fv tetramerization. This fusion protein retained a Biotin binding activity similar to natural Streptavidin as shown in Ouchterlony assay and ELISA. Death receptor 4 antigen binding activity of purified hAY4 single-chain Fv fused to Streptavidin was also confirmed by ELISA and Westernblot. In addition, surface plasmon resonance analysis showed 60-fold higher antigen binding affinity of the hAY4-STR than monomeric hAY4 ScFv due to tetramerization. In summary, hAY4 single-chain Fv fused to Streptavidin fusion protein was successfully expressed and purified as a soluble tetramer in E. coli and showed both Biotin and DR4 antigen binding activity suggesting possible production of bifunctional and tetrameric ScFv antibody.
Keywords
single-chain Fv; single-chain Fv fused to Streptavidin; streptavidin; tetramerization;
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