| Literature DB >> 12665803 |
Yoko Yamamoto1, Yasuo Tsutsumi, Yasuo Yoshioka, Toshihide Nishibata, Kyoko Kobayashi, Takayuki Okamoto, Yohei Mukai, Tomoe Shimizu, Shinsaku Nakagawa, Satoshi Nagata, Tadanori Mayumi.
Abstract
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-alpha (TNF-alpha). We prepared phage libraries expressing TNF-alpha mutants in which all the lysine residues were replaced with other amino acids. A fully bioactive lysine-deficient mutant TNF-alpha (mTNF-alpha-Lys(-)) was isolated by panning against TNF-alpha-neutralizing antibody despite reports that some lysine residues were essential for its bioactivity. mTNF-alpha-Lys(-) was site-specifically mono-PEGylated at its N terminus. This mono-PEGylated mTNF-alpha-Lys(-), with superior molecular uniformity, showed higher bioactivity in vitro and greater antitumor therapeutic potency than randomly mono-PEGylated wild-type TNF-alpha. These results suggest the usefulness of the phage display system for creating functional mutant proteins and of our site-specific PEGylation approach.Entities:
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Year: 2003 PMID： 12665803 DOI： 10.1038/nbt812
Source DB: PubMed Journal: Nat Biotechnol ISSN： 1087-0156 Impact factor: 54.908