Site-specific PEGylation of a lysine-deficient TNF-alpha with full bioactivity.
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.
Yamamoto
Yoko
Y
Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
Tsutsumi
Yasuo
Y
Yoshioka
Yasuo
Y
Nishibata
Toshihide
T
Kobayashi
Kyoko
K
Okamoto
Takayuki
T
Mukai
Yohei
Y
Shimizu
Tomoe
T
Nakagawa
Shinsaku
S
Nagata
Satoshi
S
Mayumi
Tadanori
T
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
D046911:Macromolecular Substances; D019151:Peptide Library; D014409:Tumor Necrosis Factor-alpha; D011092:Polyethylene Glycols; D008239:Lysine