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

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Transient transfection is crucial for protein expression but often suffers from uncontrolled overexpression, leading to artifacts and confounding functional analyses.
• Existing methods for controlling protein abundance, such as DNA titration or promoter swapping, offer limited and inconsistent regulation.
• Generating stable cell lines for multiple protein variants is often impractical for researchers.

Purpose of the Study:

• To develop a modular strategy using human 5' untranslated regions (5'UTRs) to achieve fine-grained and reproducible control of protein expression during transient transfection.
• To establish a versatile toolkit of 5'UTRs that can modulate protein abundance across a dynamic range for diverse protein types.
• To demonstrate the utility of tunable protein expression for improving functional analyses and experimental fidelity.

Main Methods:

• A panel of ten human 5' untranslated regions (5'UTRs) was identified and tested for their ability to regulate protein expression.
• The efficacy of these 5'UTRs was evaluated across six different proteins, including soluble and membrane proteins (e.g., eYFP, TRPA1, TRPV1, TRPM8).
• The impact of 5'UTR-mediated expression tuning on specific experimental assays, such as proximity biotinylation and stress granule marker specificity, was assessed.

Main Results:

• The panel of 5'UTRs provided a reproducible dynamic range of protein expression, enabling fine-grained control across different protein types.
• One specific 5'UTR consistently suppressed expression, effectively alleviating overexpression artifacts and improving the functional analysis of a hyperactive channel variant.
• Other 5'UTRs enhanced the expression of sensory receptors like TRPV1 and TRPM8, increasing protein yield in heterologous systems.
• The 5'UTR toolkit demonstrated superior performance compared to promoter swapping for continuous tuning of expression levels.

Conclusions:

• Human 5'UTRs represent a compact, versatile, and broadly applicable tool for fine-tuning protein abundance in transient transfection.
• This strategy enables more physiologically relevant protein expression levels, optimizing experiments and reducing artifacts.
• The 5'UTR toolkit enhances experimental fidelity by allowing researchers to match protein abundance to specific assay requirements.