Quantitative Immunocytochemical Staining for Recombinant Tissue-Type Plasminogen Activator in Transfected Chinese Hamster Ovary Cells

Abstract

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.

Constitutive Overexpression of Secreted Heterologous Proteins Decreases Extractable BiP and Protein Disulfide Isomerase Levels in Saccharomyces cerevisiae

“…For example, expression of the hepatitis B large surface protein in S. cereuisiae leads to retention in the ER and causes enlargement of the ER cisternae (Biemans et al, 1991). Immunolocalization of secreted foreign or mutant proteins in yeast cells (Bielefeld & Hollenberg, 1989) and CHO cells (Gennaro et al, 1991) shows enlarged ER cisternae, also indicating that exit from the ER is ratelimiting.…”

The cytoplasmic domain of FcγRIIA (CD32) participates in phagolysosome formation

“…Acid phosphatase is an enzyme specific for lysosomes and has been used extensively to stain CHO cells. 20 Therefore, we used this enzyme to detect the localization of lysosomal enzymes inside cells. After incubation of transfectants expressing either wild-type Fc␥RIIA or tailless Fc␥RIIA in the presence of CR3 with IgG-coated sheep erythrocytes, the cells were fixed and stained for acid phosphatase.…”

Effect of serum-free and serum-containing medium on cellular levels of ER-based proteins in various mouse hybridoma cell lines

“…Although a high level of gene transcription is attainable, the secretion of the corresponding proteins often does not correlate with this increase (Dorner and Kaufman, 1990). It could be established for many cases that retention of the synthetized polypeptides within the endoplasmic reticulum (ER) appears to be responsible for this bottleneck (Gennaro et al, 1991).…”