Improving the production of recombinant human bone morphogenetic protein-4 in Chinese hamster ovary cell cultures by inhibition of undesirable endocytosis
Abstract
Endocytic regulation serves a critical role in modulating the extracellular level of signaling molecules, such as bone morphogenetic proteins (BMPs). Unfortunately, endocytosis may result in poor yields of recombinant human BMP-4 (rhBMP-4) from Chinese hamster ovary (CHO) cell cultures. When rhBMP-4 was incubated with CHO cells, rhBMP-4 was actively internalized into cells. Cell surface bound heparan sulfate proteoglycans (HSPGs) served as the major receptors for rhBMP-4 internalization. Removal of cell surface heparan sulfate (HS) by heparinases or reduction of HSPG synthesis by knockdown of xylosyltransferase2 (xylt2) in CHO cells decreased internalization of rhBMP-4. In addition, treatment with endocytosis inhibitors (chlorpromazine, genistein, and dynasore) identified a clathrin- and dynamin-dependent endocytic pathway as the major route for rhBMP-4 internalization. To enhance product yield by minimizing rhBMP-4 internalization in recombinant CHO (rCHO) cell cultures, we have tested various strategies to reduce HSPG synthesis (knockdown of xylt2 and sodium chlorate treatment) or inhibit the binding of rhBMP-4 to cell-surface-bound HSPGs (supplementation with heparin or dextran sulfate [DS]). Among these approaches, DS, whic...Continue Reading
References
Endogenous heparan sulfate and heparin modulate bone morphogenetic protein-4 signaling and activity.
Heparan sulfate proteoglycans mediate internalization and propagation of specific proteopathic seeds
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