Nomura Tetsuya, Abe Yasuhiro, Kamada Haruhiko, Inoue Masaki, Kawara Tomoyuki, Arita Shuhei, Furuya Takeshi, Yoshioka Yasuo, Shibata Hiroko, Kayamuro Hiroyuki, Yamashita Takuya, Nagano Kazuya, Yoshikawa Tomoaki, Mukai Yohei, Nakagawa Shinsaku, Taniai Madoka, Ohta Tsunetaka, Tsunoda Shin-ichi, Tsutsumi Yasuo
National Institute of Biomedical Innovation (NiBio), 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan.
Biochem Biophys Res Commun. 2009 Oct 30;388(4):667-71. doi: 10.1016/j.bbrc.2009.08.052. Epub 2009 Aug 13.
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.
肿瘤坏死因子（TNF）通过其受体TNFR1和TNFR2发挥作用，在宿主防御和预防肿瘤形成中起着重要作用，但其受体的功能尚不清楚。为此，我们一直使用噬菌体展示技术分离TNF受体选择性突变体。然而，由于大肠杆菌的转化效率有限，难以构建具有大量库容量（>10^8）的噬菌体文库。因此，目前很难创建一个包含七个以上残基氨基酸替代的突变体文库。为了克服这个问题，我们在这里使用了两个不同的TNF突变体文库，每个文库在六个选定的氨基酸残基处含有随机替代，并利用基因改组方法构建了一个在TNF的12个不同氨基酸残基处含有替代的随机突变体文库。因此，使用这个文库，我们鉴定出了比现有突变体具有更高受体选择性和增强受体特异性生物活性的TNF突变体。
