AUTHOR=Fages-Lartaud Maxime , Tietze Lisa , Elie Florence , Lale Rahmi , Hohmann-Marriott Martin Frank 

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

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=10

YEAR=2022

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2022.892138

DOI=10.3389/fbioe.2022.892138

ISSN=2296-4185

ABSTRACT=<p>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 <italic>mCherry</italic> variant that lacks background expression as an improved tool for imaging and protein expression studies.</p>