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mCherry contains a fluorescent protein isoform that interferes with its reporter function
Maxime Fages-Lartaud1, Lisa Tietze1, Florence Elie1
1Department of Biotechnology, Norwegian University of Science and Technology, Trondheim, Norway.
Abstract:
Fluorescent proteins are essential reporters in cell and molecular biology. Here, we found that red-fluorescent proteins possess an alternative translation initiation site that produces a short functional protein isoform in both prokaryotes and eukaryotes. The short isoform creates significant background fluorescence that biases the outcome of expression studies. In this study, we identified the short protein isoform, traced its origin, and determined the extent of the issue within the family of red fluorescent protein. Our analysis showed that the short isoform defect of the red fluorescent protein family may affect the interpretation of many published studies. We provided a re-engineered mCherry variant that lacks background expression as an improved tool for imaging and protein expression studies.
Insights
Red fluorescent proteins have a hidden short form that causes background noise, affecting research. A new mCherry variant eliminates this issue for better cell biology studies.
Area of Science:
- Cell and molecular biology
- Biochemistry
- Genetics
Background:
- Fluorescent proteins are vital tools for tracking biological processes.
- Red fluorescent proteins (RFPs) are widely used but can be affected by an uncharacterized short isoform.
- This short isoform may introduce background fluorescence, potentially skewing experimental results.
Purpose of the Study:
- To identify the short functional protein isoform in red fluorescent proteins.
- To investigate the origin and prevalence of this short isoform.
- To assess the impact of this isoform on published expression studies and develop an improved reporter.
Main Methods:
- Bioinformatic analysis to identify alternative translation initiation sites.
- In vitro and in vivo expression studies in prokaryotic and eukaryotic systems.
- Comparative analysis of wild-type and engineered red fluorescent protein variants.
Main Results:
- An alternative translation initiation site was identified in red fluorescent proteins, producing a short functional isoform.
- This short isoform was detected in both prokaryotic and eukaryotic expression systems.
- The presence of the short isoform leads to significant background fluorescence, potentially affecting data interpretation in numerous studies.
- A re-engineered mCherry variant was developed, demonstrating the absence of background expression.
Conclusions:
- The short isoform of red fluorescent proteins represents a significant artifact that can compromise the accuracy of expression studies.
- This finding necessitates a re-evaluation of previous studies utilizing red fluorescent proteins.
- The engineered mCherry variant offers a more reliable tool for sensitive imaging and protein expression analysis.
