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 10 human 5' untranslated regions (5'UTRs) as a modular strategy to tune protein expression during transient transfection. Across three soluble proteins and three membrane proteins, these 5'UTRs produce a reproducible dynamic range of expression, including fine-grained control of eYFP and the large sensory ion channel TRPA1. Notably, one 5'UTR 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 5'UTRs enhance expression of the TRPV1 and TRPM8 sensory receptors, improving protein yield in heterologous systems. Together, this work identifies 5'UTRs 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.