Shimomura T, Honda T, Oouchi C, Kondo J, Nagaike K
Biosciences Laboratory, Mitsubishi Kasei Corp., Yokohama, Japan.
Cytotechnology. 1991 May;6(1):1-11. doi: 10.1007/BF00353697.
The recombinant human apolipoprotein E (Apo-E) produced by Chinese hamster ovary cells (CHO-322 cells) in serum free culture was degraded to 24K and 23K fragments that contained N-terminal amino acid. The degradation site of Apo-E to 24K fragment was between Arg180 and Leu181 and the C-terminal amino acid of 23K fragment was Gly169. In fetal bovine serum (FBS)-containing culture, the degradation was inhibited. However, in calf serum (CS) the inhibitory activity was not detected. Thus, we attempted the purification of the factor with this inhibitory activity from FBS. A protease inhibitor was purified to give a single peak from FBS by ammonium sulfate precipitation and combination of several column chromatographies. When this FBS-derived protease inhibitor (FBS-d-PI) was added to serum-free culture of CHO-322 cells, degradation of recombinant Apo-E to the 24K and 23K fragments was dose-dependently suppressed and accumulation of intact Apo-E in culture supernatant was observed. FBS-d-PI was found to be a glycoprotein with relative molecular size of 75K daltons under reducing condition, and 85K daltons under nonreducing condition by SDS-PAGE. A complex of FBS-d-PI and a cellular protease was also detected in culture supernatant by western blot analysis using mouse monoclonal antibodies against FBS-d-PI.
中国仓鼠卵巢细胞（CHO - 322细胞）在无血清培养条件下产生的重组人载脂蛋白E（Apo - E）被降解为含有N端氨基酸的24K和23K片段。Apo - E降解为24K片段的位点在Arg180和Leu181之间，23K片段的C端氨基酸为Gly169。在含有胎牛血清（FBS）的培养条件下，降解受到抑制。然而，在小牛血清（CS）中未检测到抑制活性。因此，我们尝试从FBS中纯化具有这种抑制活性的因子。通过硫酸铵沉淀和几种柱色谱法相结合，从FBS中纯化出一种蛋白酶抑制剂，得到一个单一峰。当将这种源自FBS的蛋白酶抑制剂（FBS - d - PI）添加到CHO - 322细胞的无血清培养物中时，重组Apo - E降解为24K和23K片段的过程受到剂量依赖性抑制，并且在培养上清液中观察到完整Apo - E的积累。通过SDS - PAGE发现，FBS - d - PI在还原条件下是相对分子质量为75K道尔顿的糖蛋白，在非还原条件下为85K道尔顿。使用针对FBS - d - PI的小鼠单克隆抗体进行蛋白质印迹分析，在培养上清液中也检测到FBS - d - PI与细胞蛋白酶的复合物。
