A group of researchers from Norway found that mCherry – and possibly other fluorescen proteins – has a shorter translated isoform in bacteria. This could affect localization and expression studies.
mCherry is a widely used fluorescent protein. It is derived from DsRed – the first red fluorescent protein. DsRed is a tetramer, and it went through several lab-made evolutionary steps to make it monomeric (mRFP), brighter and other qualities.
Now, the group of Hohman-Marriott from the Norwegian University of Science and Technology report in a bioRxiv pre-print that mCherry is also translated to a shorter isoform in bacteria.
The source of this isoform stems from a modification made to mRFP1.3 – the predecessor of mCherry: the initial 7 amino acids of eGFP were added to the N-terminus of mRFP1.1, followed by a 4 amino-acids long linker, which contains a methionine. The RNA sequence upstream to this methionine contains a Shine-Dalgarno (SD) sequence. In bacteria, this is a ribosomal binding site. The combination of the methionine at position 10 with a SD site just upstream leads to translation of a shorter – but still fluorescent mCherry protein. This is a problem when the mCherry is fused at the C-terminal of a protein of interest – as shown by the researhcer, when they fused to sfGFP. Expression of the mCherry was seen even when stop codons were inserted between the sfGFP and mCherry. But the researchers show that replacing the methionine at position 10 eliminates this variant (no mCherry expression). De-optimizing the SD sequence did not eliminate this (and even got higher signal compared to the optimized mCherry). The authors don’t really dwell on this, or explain this result. It suggests that the SD is not really important here – only the methionine is.
Anyway, the researchers say that any protein derived from mRFP1.3 can have this problem (e.g. mOrange, mApple, mStrawberry, PAmCherry and many others). But also, they list other fluorescent proteins with the same eGFP-linker, such as mNeonGreen, or with a methionine in the first 10 amino acids, such as KillerRed, Dronpa, mEosEM and others.
This is a problem in bacteria – and they show it for mCherry in several species. But they have not checked it in eukaryotes, but most likely its not a problem there – but caution should be taken and it is always recommended to check (e.g. by Western blot) that the correct protein size is expressed in your system.
Maxime Fages-Lartaud, Lisa Tietze, Florence Elie, Rahmi Lale, Martin Frank Hohmann-Marriott (2021) mCherry contains a fluorescent protein isoform that interferes with its reporter function
bioRxiv 2021.12.07.471677
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