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Development of protein binders that interact specifically with a given target molecule with high affinity has been of interest for their pharmaceutical and industrial applications. For this purpose, antibodies (Abs) are the primary choice for proteins capable of specific high affinity binding to target molecules, which can be isolated by immunization or from synthetic antibody library in vitro. As an alternative to Abs, nonimmunoglobulin proteins have been extensively exploited as structural scaffold to isolate in vitro target specific high affinity binders from the synthetic library randomized particular regions on the constant scaffold. Here we report a novel target-specific protein binder developed based on the scaffold of kringle domain (KD), which is present as a structual modular unit in 31 human proteins. By exploiting extensive divergence of the primary structure in the 7 loop regions, but the highly conserved backbone folding by core residues and disulfide bond linkages of the naturally occurring human KDs, we generated synthetic KD library on the yeast cell surface by randomizing the residues in the highly variable and surface exposed loops. From the KD scaffold library, we screened to isolate agonistic KD variants specfically binding to anticancer target proteins of human death receptor 4 (DR4) and/or DR5 and inducing apoptotic cell death as a single agent for several cancer cells in vitro and their in vivo tumor xenograft models. In addition to the agonists, we can also isolate from the libary an antagonistic KD variant against human tumor necrosis factor-α (TNFα), which efficiently neutralized the
TNFα-induced cytotoxicity in vitro and in vivo. The selected KD variants retained the secondary strucutre and high thermal stability, comparable to those of wild type KD, suggesting that KD scaffold are strongly seqeunce-tolerant. Our results suggest that KD scaffold can be developed as an attractive target-specific protein binder functioning as agonists or antagonists to given target molecules to modulate their biological activity.

저자

• Yong-Sung Kim [ Dept. of Molecular Science and Technology, Ajou University ]
• Chang-Han Lee [ Dept. of Molecular Science and Technology, Ajou University ]
• Kyung-Jin Park [ Dept. of Molecular Science and Technology, Ajou University ]

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• 간행물명

  한국생물공학회 학술대회

• 간기

  반년간

• 수록기간

  1985~2013

• 십진분류

  KDC 476 DDC 576