title: 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.
authors.affiliation: Department of Experimental Pathology, SmithKline Beecham Pharmaceuticals, Philadelphia, Pennsylvania 19406.
authors.forename: D E
authors.initials: DE
authors.lastname: Gennaro
authors.forename: S T
authors.initials: ST
authors.lastname: Hoffstein
authors.forename: G
authors.initials: G
authors.lastname: Marks
authors.forename: L
authors.initials: L
authors.lastname: Ramos
authors.forename: M S
authors.initials: MS
authors.lastname: Oka
authors.forename: M E
authors.initials: ME
authors.lastname: Reff
authors.forename: T K
authors.initials: TK
authors.lastname: Hart
authors.forename: P J
authors.initials: PJ
authors.lastname: Bugelski
mesh_terms: D000818:Animals; D006224:Cricetinae / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005260:Female; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D066298:In Vitro Techniques; D009685:Nuclear Envelope / Q000378:metabolism; D010053:Ovary; D011994:Recombinant Proteins / Q000032:analysis; D010959:Tissue Plasminogen Activator / Q000032:analysis*; D014162:Transfection; D014617:Vacuoles / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator