mCherry contains a fluorescent protein isoform that interferes with its reporter function

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.