Merlin S, Rowold E, Abegg A, Berglund C, Klover J, Staten N, McKearn J P, Lee S C
Searle Research and Development, Monsanto Company, St. Louis, MO 63198, USA.
Appl Biochem Biotechnol. 1997 Sep;67(3):199-214. doi: 10.1007/BF02788798.
A deletion derivative of the cytokine human interleukin-3 (hIL-3(15-125), comprising amino acids 15-125 of the native protein) was produced as a fusion to the filamentous phage surface protein pIII. The cytokine was detected in association with phage particles by protein immunoblotting. Compared to an equivalent quantity of soluble-cytokine, phage-presented hIL-3(15-125) exhibited reduced biological activity in a hIL-3-dependent cell proliferation assay. The reduction in activity was attributable to presence of phage particles in the assay, rather than directly owing to physical incorporation of the cytokine into the phage particle. Owing to the position of the amber codon in the phagemid vector, the phagemid-produced free hIL-3(15-125) species (designated hIL-3(15-125) epsilon) had 20 amino acids appended to its C-terminus; hIL-3(15-125) epsilon did not exhibit reduced bioactivity. hIL-3(15-125)-presenting phage were affinity-selected with either a hIL-3-reactive polyclonal antibody or with cells expressing the heterodimeric hIL-3 receptor. These data are consistent with the use of phage-display technology for the affinity selection of hIL-3 variants with modified biological properties.
细胞因子人白细胞介素-3（hIL-3(15 - 125)，包含天然蛋白质的第15至125个氨基酸）的缺失衍生物被制备为与丝状噬菌体表面蛋白pIII的融合体。通过蛋白质免疫印迹法在与噬菌体颗粒相关联的情况下检测到了该细胞因子。在hIL-3依赖的细胞增殖试验中，与等量的可溶性细胞因子相比，噬菌体展示的hIL-3(15 - 125)表现出降低的生物学活性。活性降低归因于试验中存在噬菌体颗粒，而不是直接由于细胞因子物理掺入噬菌体颗粒。由于琥珀密码子在噬菌粒载体中的位置，噬菌粒产生的游离hIL-3(15 - 125)物种（命名为hIL-3(15 - 125)ε）在其C末端附加了20个氨基酸；hIL-3(15 - 125)ε没有表现出降低的生物活性。用hIL-3反应性多克隆抗体或表达异二聚体hIL-3受体的细胞对展示hIL-3(15 - 125)的噬菌体进行亲和选择。这些数据与使用噬菌体展示技术进行具有修饰生物学特性的hIL-3变体的亲和选择是一致的。
