Buxser S, Vroegop S, Decker D, Hinzmann J, Poorman R, Thomsen D R, Stier M, Abraham I, Greenberg B D, Hatzenbuhler N T
Department of Cell Biology, Upjohn Company, Kalamazoo, Michigan 49001.
J Neurochem. 1991 Mar;56(3):1012-8. doi: 10.1111/j.1471-4159.1991.tb02022.x.
Human nerve growth factor (NGF) was cloned and engineered for expression in a baculovirus-infected Spodoptera frugiperda (SF-9) insect cell system. Culture supernatants contained 2-3 mg/L of recombinant human NGF. The human NGF produced by this system was purified to apparent homogeneity with a single-step affinity chromatography procedure using a high-affinity monoclonal antibody originally raised against murine NGF. The purification procedure yielded 1-2 mg of pure, human NGF per liter of culture supernatant; i.e., approximately 60% recovery of the human NGF originally released into the culture medium. Although the gene transfected into the SF-9 cells coded for pro-NGF, the NGF recovered after purification was greater than 95% fully processed, mature protein. The KD for the affinity of the pure, recombinant human NGF for NGF receptor in PC12 membranes is 0.20 +/- 0.05 nM. Activation of neurite outgrowth in PC12 cells occurs with ED50 values of 85 +/- 20 pM and 9.6 +/- 1.5 pM for a 3-day primary response and a 1-day secondary response, respectively. The pure, recombinant human NGF also stimulates a significant increase in dopamine content of PC12 cells with an ED50 of 5.8 +/- 2.7 pM. These binding and biological activation properties are consistent with values observed using murine NGF purified from submaxillary glands.
人神经生长因子（NGF）被克隆并进行基因工程改造，以便在杆状病毒感染的草地贪夜蛾（SF - 9）昆虫细胞系统中表达。培养上清液中含有2 - 3毫克/升的重组人NGF。利用最初针对鼠NGF产生的高亲和力单克隆抗体，通过单步亲和层析程序将该系统产生的人NGF纯化至表观均一性。纯化程序每升培养上清液可得到1 - 2毫克纯的人NGF；即，最初释放到培养基中的人NGF回收率约为60%。尽管转染到SF - 9细胞中的基因编码的是前体NGF，但纯化后回收的NGF超过95%是完全加工成熟的蛋白质。纯化的重组人NGF与PC12细胞膜中NGF受体的亲和力KD为0.20±0.05纳摩尔。PC12细胞中神经突生长的激活，对于3天的初级反应和1天的次级反应，ED50值分别为85±20皮摩尔和9.6±1.5皮摩尔。纯化的重组人NGF还能刺激PC12细胞多巴胺含量显著增加，ED50为5.8±2.7皮摩尔。这些结合和生物激活特性与使用从颌下腺纯化的鼠NGF所观察到的值一致。
