title: Novel protein engineering strategy for creating highly receptor-selective mutant TNFs.
abstract: Tumor necrosis factor (TNF) plays important roles in host defense and in preventing tumor formation by acting via its receptors, TNFR1 and TNFR2, functions of which are less understood. To this end, we have been isolating TNF receptor-selective mutants using phage display technique. However, generation of a phage library with large repertoire (>10(8)) is impeded by the limited transformation efficiency of Escherichia coli. Therefore, it is currently difficult to create a mutant library containing amino acid substitutions in more than seven residues. To overcome this problem, here we have used two different TNF mutant libraries, each containing random substitutions at six selected amino acid residues, and utilized a gene shuffling method to construct a randomized mutant library containing substitutions at 12 different amino acid residues of TNF. Consequently, using this library, we identified TNF mutants with greater receptor-selectivity and enhanced receptor-specific bioactivity than the existing mutants.
authors.affiliation: National Institute of Biomedical Innovation (NiBio), 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Nomura
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Abe
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Kamada
authors.forename: Masaki
authors.initials: M
authors.lastname: Inoue
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Kawara
authors.forename: Shuhei
authors.initials: S
authors.lastname: Arita
authors.forename: Takeshi
authors.initials: T
authors.lastname: Furuya
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Yoshioka
authors.forename: Hiroko
authors.initials: H
authors.lastname: Shibata
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kayamuro
authors.forename: Takuya
authors.initials: T
authors.lastname: Yamashita
authors.forename: Kazuya
authors.initials: K
authors.lastname: Nagano
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Yoshikawa
authors.forename: Yohei
authors.initials: Y
authors.lastname: Mukai
authors.forename: Shinsaku
authors.initials: S
authors.lastname: Nakagawa
authors.forename: Madoka
authors.initials: M
authors.lastname: Taniai
authors.forename: Tsunetaka
authors.initials: T
authors.lastname: Ohta
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Tsunoda
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Tsutsumi
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D032621:DNA Shuffling; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D019151:Peptide Library; D015202:Protein Engineering / Q000379:methods*; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000819:agonists* / Q000378:metabolism; D047889:Receptors, Tumor Necrosis Factor, Type II / Q000819:agonists* / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D019151:Peptide Library; D047888:Receptors, Tumor Necrosis Factor, Type I; D047889:Receptors, Tumor Necrosis Factor, Type II; D014409:Tumor Necrosis Factor-alpha