22 DHFR coamplification of T-PA in DHFR positive bovine endothelial cells: characterization of the purified serine protease

Connors, Richard W.; Sweet, Raymond W.; Noveral, James P.; Pfarr, David S.; Trill, John J.; Shebuski, Ronald J.; Berkowitz, Barry A.; Williams, D.; Franklin, S.T.; Reff, Mitchell E.

doi:10.1016/0268-9499(88)90047-1

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“…CHO cells were transfected with the human gene for tPA in an expression vector similar to that described previously for bovine endothelial cells (12) and selected in methotrexate for high level tPA secretion. The recombinant CHO cells were maintained as suspension cultures ( 5 % COZ, 95% air, 37°C) in spinner flasks in minimal essential medium (Gibco, Grand Island, NY) supplemented with 5 % fetal bovine serum and 200 n,M methotrexate.…”

Section: Methodsmentioning

confidence: 99%

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

Gennaro¹,

Hoffstein²,

Marks³

et al. 1991

Experimental Biology and Medicine

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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.

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Section: Methodsmentioning

confidence: 99%