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Site-specific PEGylation of a lysine-deficient TNF-α with full bioactivity

Nature Biotechnology volume 21, pages 546552 (2003) | Download Citation



Addition of polyethylene glycol to protein (PEGylation) to improve stability and other characteristics is mostly nonspecific and may occur at all lysine residues, some of which may be within or near an active site. Resultant PEGylated proteins are heterogeneous and can show markedly lower bioactivity. We attempted to develop a strategy for site-specific mono-PEGylation using tumor necrosis factor-α (TNF-α). We prepared phage libraries expressing TNF-α mutants in which all the lysine residues were replaced with other amino acids. A fully bioactive lysine-deficient mutant TNF-α (mTNF-α-Lys(−)) was isolated by panning against TNF-α-neutralizing antibody despite reports that some lysine residues were essential for its bioactivity. mTNF-α-Lys(−) was site-specifically mono-PEGylated at its N terminus. This mono-PEGylated mTNF-α-Lys(−), with superior molecular uniformity, showed higher bioactivity in vitro and greater antitumor therapeutic potency than randomly mono-PEGylated wild-type TNF-α. These results suggest the usefulness of the phage display system for creating functional mutant proteins and of our site-specific PEGylation approach.

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This study was supported in part by a Grant-in-Aid for Scientific Research (No. 13672385) from the Ministry of Education, Science and Culture of Japan, and in part by Health Sciences Research Grants for Research on Health Sciences focusing on Drug Innovation from the Japan Health Sciences Foundation (KH63124). We thank Ira Pastan (US National Cancer Institute, National Institutes of Health) for advice and discussion, and Kenneth Santora (a member of Ira Pastan's lab at NCI/NIH) for editorial assistance.

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Author notes

    • Yoko Yamamoto
    •  & Yasuo Tsutsumi

    These authors contributed equally to the work.


  1. Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.

    • Yoko Yamamoto
    • , Yasuo Tsutsumi
    • , Yasuo Yoshioka
    • , Toshihide Nishibata
    • , Kyoko Kobayashi
    • , Takayuki Okamoto
    • , Yohei Mukai
    • , Tomoe Shimizu
    • , Shinsaku Nakagawa
    •  & Tadanori Mayumi
  2. Laboratory of Molecular Biology, Division of Basic Science, National Cancer Institute, National Institutes of Health, Building 37, Room 4E16, 37 Convent Drive MSC 4255, Bethesda, MD 20892-4255, USA.

    • Satoshi Nagata


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The authors declare no competing financial interests.

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Correspondence to Yasuo Tsutsumi.

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