Constitutively active Rheb mutants [T23M] and [E40K] drive increased production and secretion of recombinant protein in Chinese hamster ovary cells. Issue 7 (14th April 2021)

• Record Type: Journal Article
• Authors: De Poi, Stuart P.; Xie, Jianling; Smales, C. Mark; Proud, Christopher G.
• Is Part Of: Biotechnology and bioengineering. Volume 118:Issue 7(2021)
• Page Start: 2422
• Page End: 2434
• Publication Date: 2021-04-14
• Subjects: biologic drug -- defined medium -- mTORC1 -- recombinant proteins -- Rheb; Biotechnology -- Periodicals; Bioengineering -- Periodicals; 660.6
• DOI: 10.1002/bit.27748
• Languages: English
• ISSNs: 0006-3592

Abstract

Abstract: Monoclonal antibodies (mAbs) are high value agents used for disease therapy ("biologic drugs") or as diagnostic tools which are widely used in the healthcare sector. They are generally manufactured in mammalian cells, in particular Chinese hamster ovary (CHO) cells cultured in defined media, and are harvested from the medium. Rheb is a small GTPase which, when bound to GTP, activates mechanistic target of rapamycin complex 1, a protein kinase that drives anabolic processes including protein synthesis and ribosome biogenesis. Here, we show that certain constitutively active mutants of Rheb drive faster protein synthesis in CHO cells and increase the expression of proteins involved in the processing of secreted proteins in the endoplasmic reticulum, which expands in response to expression of Rheb mutants. Active Rheb mutants, in particular Rheb[T23M], drive increased cell number under serum‐free conditions similar to those used in the biotechnology industry. Rheb[T23M] also enhances the expression of the reporter protein luciferase and, especially strongly, the secreted Gaussia luciferase. Moreover, Rheb[T23M] markedly (2–3 fold) enhances the amount of this luciferase and of a model immunoglobulin secreted into the medium. Our data clearly demonstrate that expressing Rheb[T23M] in CHO cells provides a simple approach to promoting their growth in defined medium and the production of secreted proteins of high commercial value. Abstract : Constitutively active Rheb mutants (Rheb*) activate mTORC1 signaling which in turn drives multiple anabolic processes including protein synthesis and ribosome biogenesis. Combined with increased cell growth and number, this increases protein production. Likely as a consequence of this, the UPR is triggered, resulting in enhanced cellular capacity for protein folding and processing. Overall, there is a marked increase in the output of secreted protein(s).