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Constitutively-active Rheb mutants [T23M] and [E40K] drive increased production and secretion of recombinant protein in Chinese hamster ovary cells

De Poi, Stuart P, Xie, Jianling, Smales, Christopher Mark, Proud, Christopher G (2021) Constitutively-active Rheb mutants [T23M] and [E40K] drive increased production and secretion of recombinant protein in Chinese hamster ovary cells. Biotechnology and Bioengineering, 118 (7). pp. 2422-2434. ISSN 0006-3592. (doi:10.1002/bit.27748) (KAR id:89632)

Abstract

Monoclonal antibodies (mAbs) 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 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.

Item Type: Article
DOI/Identification number: 10.1002/bit.27748
Subjects: Q Science
Divisions: Divisions > Division of Natural Sciences > Biosciences
Depositing User: Mark Smales
Date Deposited: 05 Aug 2021 10:04 UTC
Last Modified: 14 Nov 2022 23:13 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/89632 (The current URI for this page, for reference purposes)

University of Kent Author Information

Smales, Christopher Mark.

Creator's ORCID: https://orcid.org/0000-0002-2762-4724
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