Cabibbo A, Sporeno E, Toniatti C, Altamura S, Savino R, Paonessa G, Ciliberto G
Department of Genetics, Istituto di Ricerche di Biologia Moleculare P. Angeletti (IRBM), Pomezìa (Roma), Italy.
Gene. 1995 Dec 29;167(1-2):41-7. doi: 10.1016/0378-1119(95)00632-x.
Phage display of proteins can be used to study ligand-receptor interaction and for the affinity-maturation of binding sites in polypeptide hormones and/or cytokines. We have expressed human interleukin-6 (hIL-6) on M13 phage in a monovalent fashion as a fusion protein with the phage coat protein, pIII. Phage-displayed hIL-6 is correctly folded, as judged by its ability to interact with conformation-specific anti-hIL-6 monoclonal antibodies (mAb) and with the hIL-6 receptor complex in vitro. We set up an experimental protocol for the efficient affinity selection of hIL-6 phage using the extracellular portion of the hIL-6 receptor alpha (hIL-6R alpha) fixed on a solid phase. This system was used to affinity-purify from a library of hIL-6 variants, in which four residues in the predicted D-helix of the cytokine were fully randomized, mutants binding hIL-6R alpha with higher efficiency than the wild type. When the best-binder variant Q175I/Q183A was combined with a previously identified superbinder S176R [Savino et al., Proc. Natl. Acad. Sci. 90 (1993) 4067-4071], a triple-substitution mutant Q175I/S176R/Q183A (hIL-6IRA) was obtained with a fivefold increased hIL-6R alpha binding and a 2.5-fold enhanced biological activity.
蛋白质的噬菌体展示可用于研究配体 - 受体相互作用以及多肽激素和/或细胞因子中结合位点的亲和力成熟。我们已将人白细胞介素 - 6（hIL - 6）以单价形式作为与噬菌体外壳蛋白pIII的融合蛋白在M13噬菌体上表达。通过其在体外与构象特异性抗hIL - 6单克隆抗体（mAb）以及hIL - 6受体复合物相互作用的能力判断，噬菌体展示的hIL - 6折叠正确。我们建立了一种实验方案，用于使用固定在固相上的hIL - 6受体α（hIL - 6Rα）的细胞外部分对hIL - 6噬菌体进行高效亲和力选择。该系统用于从hIL - 6变体文库中进行亲和纯化，其中细胞因子预测的D - 螺旋中的四个残基完全随机化，与野生型相比，突变体与hIL - 6Rα结合效率更高。当最佳结合变体Q175I/Q183A与先前鉴定的超级结合体S176R [Savino等人，美国国家科学院院刊90（1）（1993）4067 - 4071]结合时，获得了三取代突变体Q175I/S176R/Q183A（hIL - 6IRA），其与hIL - 6Rα的结合增加了五倍，生物活性增强了2.5倍。
