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Abstract Mammalian cells process information through coordinated spatiotemporal regulation of proteins. Engineering cellular networks thus relies on efficient tools for regulating protein levels in specific subcellular compartments. To address the need to manipulate the extent and dynamics of protein localization, we developed a platform technology for the target-specific control of protein destination. This platform is based on bifunctional molecules comprising a target-specific nanobody and universal sequences determining target subcellular localization or degradation rate. We demonstrate that nanobody-mediated localization depends on the expression level of the target and the nanobody, and the extent of target subcellular localization can be regulated by combining multiple target-specific nanobodies with distinct localization or degradation sequences. We also show that this platform for nanobody-mediated target localization and degradation can be regulated transcriptionally and integrated within orthogonal genetic circuits to achieve the desired temporal control over spatial regulation of target proteins. The platform reported in this study provides an innovative tool to control protein subcellular localization, which will be useful to investigate protein function and regulate large synthetic gene circuits. 

Abstract The production of high‐quality recombinant proteins is critical to maintaining a continuous supply of biopharmaceuticals, such as therapeutic antibodies. Engineering mammalian cell factories presents a number of limitations typically associated with the proteotoxic stress induced upon aberrant accumulation of off‐pathway protein folding intermediates, which eventually culminate in the induction of apoptosis. In this review, we will discuss advances in cell engineering and their applications at different hierarchical levels of control of the expression of recombinant proteins, from transcription and translational to posttranslational modifications and subcellular trafficking. We also highlight challenges and unique opportunities to apply modern synthetic biology tools to the design of programmable cell factories for improved biomanufacturing of therapeutic proteins.