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