title: Site-specific PEGylation of a lysine-deficient TNF-alpha with full bioactivity.
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.
authors.affiliation: Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Yamamoto
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Tsutsumi
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Yoshioka
authors.forename: Toshihide
authors.initials: T
authors.lastname: Nishibata
authors.forename: Kyoko
authors.initials: K
authors.lastname: Kobayashi
authors.forename: Takayuki
authors.initials: T
authors.lastname: Okamoto
authors.forename: Yohei
authors.initials: Y
authors.lastname: Mukai
authors.forename: Tomoe
authors.initials: T
authors.lastname: Shimizu
authors.forename: Shinsaku
authors.initials: S
authors.lastname: Nakagawa
authors.forename: Satoshi
authors.initials: S
authors.lastname: Nagata
authors.forename: Tadanori
authors.initials: T
authors.lastname: Mayumi
mesh_terms: D000818:Animals; D041702:BALB 3T3 Cells; D001665:Binding Sites; D005260:Female; D005354:Fibrosarcoma / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000737:chemistry / Q000172:deficiency; D046911:Macromolecular Substances; D051379:Mice; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library; D011092:Polyethylene Glycols / Q000737:chemistry*; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D046911:Macromolecular Substances; D019151:Peptide Library; D014409:Tumor Necrosis Factor-alpha; D011092:Polyethylene Glycols; D008239:Lysine