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

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.

Optimising expression of the large dynamic range FRET pair mNeonGreen and superfolder mTurquoise2ox for use in the Escherichia coli cytoplasm

“…Coinciding with our observation of the expression of mNG from a rudimental methionine at the end of a GFP-derived N-terminus, it was also described for mCherry in a preprint by Fages-Lartaud and colleagues 30 . These scientists used an E. coli optimised version of mCh, which contained a similar RBS upstream of Met10.…”

“…Another concern we wish to address is that it is often (almost) undecipherable from publications whether fluorescent proteins have been codon-optimized and to what extent. The amino acid sequence and, therefore the resulting protein may be the same, but as this study and the work of Fages-Lartaud and colleagues 30 shows: it does matter and should be reported more clearly. sfTq2 ox and sfTq2 oxopt have the same amino acid sequence and can therefore consider to be the same protein: however, their expression in E. coli differs hugely.…”

Standard Intein Gene Expression Ramps (SIGER) for Protein-Independent Expression Control

“…Alternative translative start sites are a common issue occurring with N-terminal protein tags and fluorescent proteins. 66,68 This result support the hypothesis of an alternative start codon close to the gene start producing a GFP isoform. Nevertheless, this artifact does not interfere with the conclusion regarding the in vivo cleavage performance of DnaB-GFP.…”

“…For DnaB-mCherry , a first observation is that fluorescence levels are generally higher than expected from the DnaB-GFP range, although the relative strength ranking of GES remained the same (Figure a). This slight mCherry overexpression could be due to the newly identified alternative translation start site of mCherry that produces a short functional protein isoform . The short mCherry isoform produces significant background fluorescence when the reporter is used as a C-terminal fusion partner.…”

“…This slight mCherry overexpression could be due to the newly identified alternative translation start site of mCherry that produces a short functional protein isoform. 66 The short mCherry isoform produces significant background fluorescence when the reporter is used as a C-terminal fusion partner. Although the background fluorescence is supposedly the same across strains, the production of the short mCherry isoform blurs the relative differences in the expression range.…”