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Engineering of a human kringle domain into agonistic and antagonistic binding proteins functioning in vitro and in vivo.

Lee, CH; Park, KJ; Sung, ES; Kim, A; Choi, JD; Kim, JS; Kim, SH; Kwon, MH; Kim, YS
Proceedings of the National Academy of Sciences of the United States of America, 107(21):9567-9571, 2010
Journal Title
Proceedings of the National Academy of Sciences of the United States of America
Here, we report the development of target-specific binding proteins based on the kringle domain (KD) ( approximately 80 residues), a ubiquitous modular structural unit occurring across eukaryotic species. By exploiting the highly conserved backbone folding by core residues, but using extensive sequence variations in the seven loop regions of naturally occurring human KDs, we generated a synthetic KD library on the yeast cell surface by randomizing 45 residues in the loops of a human KD template. We isolated KD variants that specifically bind to anticancer target proteins, such as human death receptor 4 (DR4) and/or DR5, and that function as agonists to induce apoptotic cell death in several cancer cell lines in vitro and inhibit tumor progression in mouse models. Combined treatments with KD variants possessing different recognition sites on the same target protein exerted synergisitic tumoricidal activities, compared to treatment with individual variants. In addition to the agonists, we isolated an antagonistic KD variant that binds human tumor necrosis factor-alpha (TNFalpha) and efficiently neutralizes TNFalpha-induced cytotoxicity in vitro and in vivo. The KD scaffold with seven flexible loops protruding from the central core was strongly sequence-tolerant to mutations in the loop regions, offering a potential advantage of distinct binding sites for target recognition on the single domain. Our results suggest that the KD scaffold can be used to develop target-specific binding proteins that function as agonists or antagonists toward given target molecules, indicative of their potential use as biotherapeutics.
MeSH terms
Amino Acid SequenceAnimalsCell Line, TumorFemaleHumansKringles*MiceMice, Inbred BALB CMice, NudeModels, MolecularMolecular Sequence DataPeptide LibraryProtein BindingProtein EngineeringProtein FoldingProtein Interaction Domains and Motifs*Protein Structure, TertiaryProteins/chemistry*Proteins/geneticsProteins/metabolismSaccharomyces cerevisiae/geneticsSaccharomyces cerevisiae/metabolismSequence AlignmentXenograft Model Antitumor Assays
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Journal Papers > School of Medicine / Graduate School of Medicine > Microbiology
AJOU Authors
권, 명희
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