Proteostasis Engineering of Glutamine Synthetase for Stringent, Drug-Free Selection in Chinese Hamster Ovary Cells

The efficient selection of cell lines that produce high levels of therapeutic proteins is a major challenge in biotechnology. The glutamine synthetase (GS)-based selection system, widely used in Chinese hamster ovary (CHO) cells, is limited in its ability to isolate rare high-producing clones due to the intrinsic stability of the GS protein. This stability lowers selection stringency, allowing survival of cells with suboptimal productivity. To address this limitation, we engineered GS variants incorporating modular degrons to actively regulate protein degradation and intracellular GS levels. This strategy increases selection pressure by controlling protein stability, rather than relying on transcriptional or translational regulation. In CHO cells expressing either a reporter protein or Etanercept (ETN), destabilized GS (dGS) variants selectively enriched cell pools with markedly enhanced productivity. Molecular analysis revealed that these improvements were driven by increased per-copy mRNA abundance, rather than gene copy amplification. The top-performing dGS pool achieved an ETN titer of 870 mg/L in shake-flask batch culture and maintained stable production over 80 days of passaging. Notably, these results were obtained without chemical selection agents or extensive gene amplification. Collectively, this study demonstrates that proteostasis engineering—modulating the balance between protein synthesis and degradation—enables high-stringency, drug-free selection of high-producing cell lines. This approach has the potential to accelerate cell line development and reduce manufacturing complexity in biopharmaceutical production. • Degradable GS variants increase selection stringency by accelerating GS protein turnover. • Degradable GS selection enriches high producers via elevated per-copy transcript output. • Drug-free dGS pools achieved 870 mg/L Etanercept titer in batch culture.