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Constitutively-active Rheb mutants [T23M] and [E40K] drive increased production and secretion of recombinant protein in Chinese hamster ovary cells
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  • Stuart De Poi,
  • Jianling Xie,
  • Christopher Mark Smales,
  • Christopher Proud
Stuart De Poi
South Australian Health and Medical Research Institute
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Jianling Xie
South Australian Health and Medical Research Institute
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Christopher Mark Smales
University of Kent
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Christopher Proud
South Australian Health and Medical Research Institute
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Abstract

Monoclonal antibodies are high value agents used for disease therapy (‘biologic drugs’) or as diagnostic tools which are widely used in the health care 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 (mTORC1), 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 via the endoplasmic reticulum, which expands in response to expression of Rheb. 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 into the medium. Our data clearly demonstrate that expressing Rheb[T23M] in CHO cells provides a simple approach to promoting cell growth in defined medium and the production of secreted proteins of high commercial value

Peer review status:ACCEPTED

07 Oct 2020Submitted to Biotechnology and Bioengineering
07 Oct 2020Submission Checks Completed
07 Oct 2020Assigned to Editor
14 Oct 2020Reviewer(s) Assigned
03 Nov 2020Editorial Decision: Revise Major
03 Nov 2020Review(s) Completed, Editorial Evaluation Pending
28 Jan 20211st Revision Received
28 Jan 2021Assigned to Editor
28 Jan 2021Submission Checks Completed
05 Feb 2021Reviewer(s) Assigned
03 Mar 2021Review(s) Completed, Editorial Evaluation Pending
03 Mar 2021Editorial Decision: Revise Minor
04 Mar 20212nd Revision Received
04 Mar 2021Assigned to Editor
04 Mar 2021Submission Checks Completed
08 Mar 2021Review(s) Completed, Editorial Evaluation Pending
08 Mar 2021Editorial Decision: Accept