Schröder M, Friedl P
Technische Hochschule Darmstadt, Institut für Biochemie, D-64287 Darmstadt, Germany. darmstadt.de
Biotechnol Bioeng. 1997 Mar 20;53(6):547-59. doi: 10.1002/(SICI)1097-0290(19970320)53:6<547::AID-BIT2>3.0.CO;2-M.
Overexpression of recombinant proteins in animal cells is commonly achieved by using gene amplification techniques. Gene amplified cells possess up to several thousand genes coding for the target protein. Constitutive expression of these genes leads to high levels of the corresponding mRNA species and the immature protein in the cell. Inefficient processing of these precursors may result from their great abundance in the cell. To study the influence of elevated intracellular levels of a recombinant protein on its maturation and secretion, we examined the maturation and secretion of human antithrombin III (hATIII) in Chinese hamster ovary (CHO) cells at different levels of gene amplification. No loss of vitality was caused by elevated secretion of hATIII. As the intracellular hATIII content increased, the efficiency of hATIII secretion decreased steadily. The state of intracellular hATIII from the different cell lines was studied by determining the specific heparin cofactor activity of hATIII. Intracellular hATIII from the highest amplified cell line displayed a lowered specific heparin cofactor activity indicating the presence of malfolded, only partially folded, or incompletely or incorrectly posttranslationally modified hATIII in this cell line. Thus, the ability of CHO cells to fold and/or introduce posttranslational modifications and subsequently to secrete the recombinant protein becomes saturated, and therefore these processes may become limiting for protein secretion at highly elevated expression levels. This limitation was not due to a general exhaustion of the secretory capacity of the cells because hATIII constituted only a minor fraction of the secreted proteins, even at high expression levels.
在动物细胞中重组蛋白的过表达通常通过基因扩增技术来实现。基因扩增的细胞拥有多达数千个编码目标蛋白的基因。这些基因的组成型表达导致细胞中相应mRNA种类和未成熟蛋白的高水平表达。这些前体的加工效率低下可能是由于它们在细胞中大量存在所致。为了研究细胞内重组蛋白水平升高对其成熟和分泌的影响，我们检测了不同基因扩增水平下中国仓鼠卵巢（CHO）细胞中人抗凝血酶III（hATIII）的成熟和分泌情况。hATIII分泌增加并未导致细胞活力丧失。随着细胞内hATIII含量的增加，hATIII的分泌效率稳步下降。通过测定hATIII的特异性肝素辅因子活性来研究不同细胞系中细胞内hATIII的状态。来自最高扩增细胞系的细胞内hATIII显示出较低的特异性肝素辅因子活性，表明该细胞系中存在错误折叠、仅部分折叠或翻译后修饰不完全或不正确的hATIII。因此，CHO细胞折叠和/或进行翻译后修饰并随后分泌重组蛋白的能力达到饱和，因此在高表达水平时这些过程可能成为蛋白质分泌的限制因素。这种限制并非由于细胞分泌能力的普遍耗尽，因为即使在高表达水平下，hATIII也仅占分泌蛋白的一小部分。
