van Erp R, Adorf M, van Sommeren A P, Gribnau T C
Clinical Lab Systems Research Unit, Organon Teknika B.V., Boxtel, The Netherlands.
J Biotechnol. 1991 Oct;20(3):249-61. doi: 10.1016/0168-1656(91)90298-a.
An acid proteolytic activity has been found in cell culture supernatants from long-term cultivations of hybridoma cells in hollow fibre bioreactors using serum free medium. The proteolytic activity has now been further characterized and the main results were: (1) the proteolytic activity showed a maximum around pH 3 and declined essentially to zero at pH 8; (2) the activity was specifically inhibited by pepstatin A; (3) the acid proteases consisted of two sets of closely spaced bands with apparent molecular weights of 40-45K and 90-105K, respectively; (4) the protease bands (40-45K and 90-105K) were reactive with anti-human cathepsin D; (5) the IEP values of the acid proteases ranged from pH 4.55-6.5. Furthermore, IgG incubation with the acid proteases isolated from hybridoma cells yielded fragments similar to those found in serum-free hollow fibre cell culture supernatants. These results indicated that the IgG fragments are the result of degradation by cathepsin D like proteases released after cell death or cell lysis.
在使用无血清培养基的中空纤维生物反应器中，对杂交瘤细胞进行长期培养时，在细胞培养上清液中发现了一种酸性蛋白水解活性。目前已对该蛋白水解活性进行了进一步表征，主要结果如下：(1)蛋白水解活性在pH 3左右达到最大值，在pH 8时基本降至零；(2)该活性被胃蛋白酶抑制剂A特异性抑制；(3)酸性蛋白酶由两组紧密间隔的条带组成，表观分子量分别为40 - 45K和90 - 105K；(4)蛋白酶条带(40 - 45K和90 - 105K)与抗人组织蛋白酶D反应；(5)酸性蛋白酶的等电点值范围为pH 4.55 - 6.5。此外，将IgG与从杂交瘤细胞中分离出的酸性蛋白酶一起孵育，产生的片段与在无血清中空纤维细胞培养上清液中发现的片段相似。这些结果表明，IgG片段是细胞死亡或细胞裂解后释放的类似组织蛋白酶D的蛋白酶降解的结果。
