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 leads to artifacts due to uncontrolled overexpression.
• Stable cell lines are an alternative but are impractical for multiple constructs.
• Current methods for controlling protein abundance offer coarse and inconsistent regulation.

Purpose of the Study:

• To establish a panel of human 5´ untranslated regions (5´UTRs) as a modular strategy for fine-tuning protein expression during transient transfection.
• To assess the dynamic range and reproducibility of expression control across different proteins.
• To demonstrate the utility of 5´UTRs in alleviating overexpression artifacts and enhancing functional analyses.

Main Methods:

• A panel of ten human 5´UTRs was generated and tested.
• Expression levels were modulated for soluble and membrane proteins, including eYFP, TRPA1, TRPV1, and TRPM8.
• Functional assays were performed to evaluate the impact of 5´UTR-mediated expression control on protein analysis.

Main Results:

• The 5´UTRs provided a reproducible dynamic range of protein expression across various proteins.
• One specific 5´UTR consistently suppressed expression, alleviating artifacts in functional assays like proximity biotinylation and stress granule marker specificity.
• Other 5´UTRs enhanced expression of sensory receptors (TRPV1, TRPM8), improving protein yield.

Conclusions:

• Human 5´UTRs serve as a versatile and broadly applicable tool for precise control of protein abundance in transient transfection.
• This strategy enables more physiologically relevant and assay-optimized protein expression, improving the accuracy of functional studies.
• The developed 5´UTR panel offers a compact solution for researchers needing fine-grained control over protein expression levels.