Deubiquitinase-Targeting Chimera (DUBTAC)-Enhanced Mammalian Expression System for Efficient Recombinant Protein Expression

Abstract Overexpression of exogenous proteins in mammalian cells frequently saturates the endogenous folding and quality control machinery, resulting in their recognition as misfolded substrates, subsequent polyubiquitination, and rapid degradation by the 26S proteasome. This cellular bottleneck directly constrains recombinant protein yields and remains a major challenge in biopharmaceutical production. To address this limitation, we developed a deubiquitinase-targeting chimera (DUBTAC)-enhanced mammalian expression system designed to selectively stabilize recombinant proteins. In this platform, the target protein is genetically fused to cellular retinoic acid-binding protein 1 (CRABP1), while a SNAP-tagged deubiquitinase (UCHL1-SNAP) is co-expressed. Upon administration of the heterobifunctional small molecule BG-ATRA, which simultaneously engages the SNAP tag and CRABP1, the deubiquitinase is recruited in proximity to the target protein. This induced proximity enables the enzymatic removal of degradation-linked ubiquitin chains, thereby shielding the recombinant protein from proteasomal destruction and extending its intracellular half-life. We demonstrated that this system significantly increased the yields of three structurally and functionally diverse recombinant proteins─thrombopoietin (TPO), osteoprotegerin (OPG), and vitronectin (VN)─by 2.6- to 3.1-fold in HEK293F suspension cells. Importantly, the enhanced production was achieved without compromising the biological activity of the purified proteins. This DUBTAC-based approach thus offers a robust, generalizable platform for boosting recombinant protein production in mammalian expression systems.