5'Untranslated regions provide a versatile toolkit for tunable exogenous protein expression

Transient transfection is widely used for protein expression in heterologous systems, yet uncontrolled overexpression frequently introduces artifacts that confound functional analyses. Although stable cell lines can mitigate these issues, generating lines for multiple constructs or variants is often impractical. Common alternatives, such as DNA titration, altered transfection conditions, or promoter swapping, provide only coarse and inconsistent control of protein abundance. Here, we establish a panel of ten human 5 untranslated regions (5UTRs) as a modular strategy to tune protein expression during transient transfection. Across three soluble proteins and three membrane proteins, these 5UTRs produce a reproducible dynamic range of expression, including fine-grained control of eYFP and the large sensory ion channel TRPA1. Notably, one 5UTR consistently suppresses expression across all proteins tested and alleviates overexpression-associated artifacts, improving functional analysis of a hyperactive channel variant, substantially reducing background in proximity biotinylation assays, and enhancing the specificity of a stress granule marker. In contrast, most 5UTRs enhance expression of the TRPV1 and TRPM8 sensory receptors, improving protein yield in heterologous systems. Together, this work identifies 5UTRs as a compact, versatile, and broadly applicable tool to fine-tune protein abundance, enabling more physiologically relevant and assay-optimized expression in transient transfection experiments. Significance StatementO_LIProtein overexpression remains a pervasive limitation in transient transfection experiments, as commonly used strategies provide coarse, labor-intensive, and often unpredictable control over protein abundance, frequently leading to artifacts or loss of physiological relevance. C_LIO_LIA compact, modular 5UTR toolkit enables fine-grained and scalable control of protein abundance in transient transfection, providing a reproducible dynamic range across soluble and membrane proteins outperforming promoter swapping for continuous tuning of expression levels. C_LIO_LITunable expression improves experimental fidelity across diverse applications, allowing investigators to match protein abundance to specific assay needs, reducing overexpression artifacts, preserving physiological readouts, and enhancing yield when high expression is required. C_LI