Gennaro D E, Hoffstein S T, Marks G, Ramos L, Oka M S, Reff M E, Hart T K, Bugelski P J
Department of Experimental Pathology, SmithKline Beecham Pharmaceuticals, Philadelphia, Pennsylvania 19406.
Proc Soc Exp Biol Med. 1991 Oct;198(1):591-8. doi: 10.3181/00379727-198-43294.
Tissue-type plasminogen activator (tPA) is a serine protease which cleaves plasminogen to its active form, plasmin. tPA plays a physiologic role in hemostasis, wound healing, and embryogenesis. Therapeutically, recombinant human tPA is used as a thrombolytic in myocardial infarction. Although production of therapeutic quantities of tPA in Chinese hamster ovary (CHO) cells transfected with the human gene for tPA is practical, production costs remain high. One important factor which determines the ultimate cost of tPA (or any other recombinant protein expressed in mammalian cells) is its production level on a per cell basis. We have used postembedding immunocytochemical staining with colloidal gold to study the subcellular localization of tPA in CHO cells expressing recombinant tPA (rCHO) in an effort to understand the factor(s) which might limit secretion. Staining for tPA was evaluated visually and by morphometric analysis and was specific and reproducible. Serially passaged rCHO showed no significant change in staining density over 31 serial passages. Staining density was greatest over dilated cisternae of the rough endoplasmic reticulum and nuclear envelope. Golgi stacks and large acid phosphatase-positive vacuoles (probably lysosomes) were also heavily stained. Staining of lysosomal vacuoles suggested that rCHO might be degrading nascent tPA. Incubation of rCHO with 125I-tPA showed that the cells were not internalizing tPA from the media. These results suggest that rCHO fail to secrete a portion of the tPA they synthesize and that it is degraded in lysosomes. This observation may have important implications on the choice of expression systems for efficient production of large quantities of recombinant proteins.
组织型纤溶酶原激活剂（tPA）是一种丝氨酸蛋白酶，可将纤溶酶原裂解为其活性形式纤溶酶。tPA在止血、伤口愈合和胚胎发生中发挥生理作用。在治疗上，重组人tPA被用作心肌梗死的溶栓剂。虽然用转染了人tPA基因的中国仓鼠卵巢（CHO）细胞生产治疗量的tPA是可行的，但生产成本仍然很高。决定tPA（或在哺乳动物细胞中表达的任何其他重组蛋白）最终成本的一个重要因素是其每细胞的生产水平。我们使用胶体金进行包埋后免疫细胞化学染色，以研究tPA在表达重组tPA的CHO细胞（rCHO）中的亚细胞定位，试图了解可能限制分泌的因素。通过视觉评估和形态计量分析对tPA染色进行评估，结果具有特异性和可重复性。连续传代的rCHO在31次连续传代中染色密度没有显著变化。染色密度在粗面内质网和核膜的扩张池上最大。高尔基体堆栈和大的酸性磷酸酶阳性液泡（可能是溶酶体）也被大量染色。溶酶体液泡的染色表明rCHO可能正在降解新生的tPA。用125I-tPA孵育rCHO表明细胞没有从培养基中内化tPA。这些结果表明rCHO未能分泌它们合成的一部分tPA，并且它在溶酶体中被降解。这一观察结果可能对高效生产大量重组蛋白的表达系统的选择具有重要意义。
