Any red fluorescent protein similar to GFP in RED-spectrum?

Model Organism: Worms Scientific Discussion
1

I posted this on the facebook page of C. elegans researchers, but haven’t received any answer. Please, help if you know the answer!

Considering aggregation (misfolding, ending up in LROs; my current main problem is fusion proteins aggregation - seamless fusions - although bleaching fast and cytotoxicity are other factors too, but not as horrible as misfolding and aggregation) and subcellular mislocalization: among mCherry, TagRFP, mKate2, FusionRed, Ruby, Tomato: Does anyone know which …FP behaves the most like eGFP but isn’t green (red spectrum) in C. elegans?

Just a side note from “A monomeric red fluorescent protein with low cytotoxicity”
Shemiakina et al.:…“It was fortuitous that the first fluorescent protein to be successfully cloned, Aequorea victoria green fluorescent protein (GFP), was naturally monomeric at intracellular expression levels and thus suitable for protein labeling in living cells”…“Engineered red monomers, such as mCherry and other mFruit proteins, along with mKO, TagRFP, and mKate2 are suitable fusion partners for many proteins of interest. However, these proteins usually do not perform as well as Aequorea victoria GFP derivatives in fusions, and often present issues related to abnormal subcellular protein localization, aggregation, or toxic effects”.

:-[ :’( So from 60s (GFP purified by Osamu Shimomura,…, Martin Chalfie expressed it in C. elegans, 1994) till now, (60 years!) don’t we have something similar to GFP in RED-spectrum?
Really frustrated! :’( please, help!.

Another question: does adding linker-glycin help? I have done it once for GFP and the result was good, but thinking to use it.

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We’ve spent an embarrassing amount of time testing and comparing different fluorescent proteins, and I can tell you that generally speaking, all of the red FPs are sub-optimal. None are as bright as GFP, they have very poor maturation efficiency (i.e. only a small fraction of molecules actually fluoresce), and they all aggregate to some degree. That being said, I think that mScarlet-I is the best of the bunch. Compared to the others it is significantly brighter, folds more efficiently and aggregates less (although it is still not a true monomer). That’s what we’re using to make all of our red tags these days. Our constructs are on Addgene (https://www.addgene.org/91826/).

It never hurts to include a glycine-serine-rich linker between the FP and your gene of interest. We usually use 9-10 residues.

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Thank you Dan,

I truly appreciate your kindness and supportive spirit (something worm community has been proud of decades) in taking care of my question and spending time sharing your valuable knowledge.

Two related questions:

1- Have you tried mNeptune?

2- Why do we need three introns in FPs?

Having introns increases the risks of alternative splicing and it is possible to end up with several non-functional FPs that may also add up to aggregation and extra toxicity. Plus, they may splice-alternatively with different exons of the inserted protein (I know we put introns to increase the level of the final FP expression, but considering splicing and non functional ones, this may cost toxicity too).

Thank you again,

This is Gholamali (you have supported us before too, here I had to sign in again with new login).

Regards.

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Hi Gholamali,

I tried one of the versions of mNeptune once - I think mNeptune2.5 but I would have to double check my notes. I never saw any fluorescence at all, but that might have been due to germline silencing. It would have been worth trying again, especially if you are working in a somatic tissue. I’ll also note that mScarlet-I is an orange-red FP (like DsRed, TagRFP) whereas mNeptune is “deep red” (even a little further red than mKate2). With proper filters you might be able to use them both together.

RE the introns, we always put three in our constructs because, frankly, that’s what everyone does and it wasn’t a parameter we cared to mess with. But apparently you don’t actually need three: https://www.nature.com/articles/s41598-019-45517-0

Cheers,
Dan

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Gholamali,

As you are evaluating different fluorescent proteins for your use, I highly recommend FPbase.org, it has a lot of information about fluorescent proteins, quantum yields, etc. https://www.fpbase.org/

Of note, GFP is not monomeric unless the A205 position is mutated, many, but not all of the vectors in the Fire Lab kit have that mutation, but be careful. mCherry often gets mislabeled as an aggregating tag, but that’s likely due to the fact that it is still fluorescent inside lysosomes. As far as anyone can tell when they specifically test mCherry it is has the lowest aggregation properties. It remains possible that tagging a protein with mCherry will change the aggregation of both. We find that mCherry tags are brighter and easier to follow than mScarlet, although we do not have as many mScarlet variants.

Best of luck.

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Hi Dan,

Thank you.

I read the paper and I wish there were more such a study with more promoters other than vit-2P and hsp-90P (if we think there is an interaction between the intron and the promoter), also a study to show the non-functional mCherry products (dark and no-fluorescent, but junk aggregates) due to increased number of introns.

I think that one of the sources of aggregation can be those two extra introns (or even all three), that potentially cause alternative splicing and misfolded isoforms.

Hi bdackley,

Thank you very much for insight and information and for directing me to FPbase.org which is a great resource.

My main problem is aggregation now. The brightness is not as concerning.

…“mCherry often gets mislabeled as an aggregating tag, but that’s likely due to the fact that it is still fluorescent inside lysosomes”…

In my hand it is not only in lysosomes (Lysosome: I guess according to the literature- I haven’t labeled lysosome personally yet), LROs (I use additional green and DAPI filter to distinguish them), but also these RED-FPs are diffuse cytoplasmic, and some threadlike structures, all membranes sometimes, plus huge irregular aggregation (I have tried 0.5-1ng/ul to 2, 3, 5, 10, 15, 25, 50-100 ng/ul, even higher concentrations and see them especially with higher ng/ul of the injected construct) that they don’t resemble as a subcellular organelle or a round well-shaped lysosomal structure or any structure.

Neither I see the expected structure to be brighter and sometimes it is totally missing as if the RED-FP is everywhere except where it must be.

Unfortunately, I don’t have the resource to investigate these Red-FPs vigorously and I wish a resourceful lab could investigate them more to offer it to C. elegans investigators. I will clone different current ones: Scarlet-I, Fusion-Red (has only one 5’-intron) and mNeptune under a promoter and 3’UTR that has been working for me without any attached protein to see which one gives me less aggregation.

I checked egfp, superfoldGFP, and alph-gfp, and nowGFP,… and found that mostly the position 225 is a T. I cannot find A225. There is either T225 or A226, or even A227

May I ask you which type of GFP you are using? and what is the mutation of A225?

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Hi Gholamali

The position is either A205 or A206 (in most of the Fire Lab sequences it’s 206), but I think in the mammalian derived ones it’s 205, so that’s the typical reference, but it is not position 225.

I hope that helps.

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Thank you and sorry for my mistake (it is not A225).

I checked avGFP https://www.fpbase.org/protein/avgfp/

and as you mentioned it is A206. The only question remains: what should A206 be converted to?

I would be grateful if you can clarify it more.

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I believe it is commonly changed to a K to create monomer GFP