After working with a set of firefly/beetle luciferases of different thermostability in mammalian cells (including the regular luc2 for Photinus pyralis and its thermostable mutants), my impression is that the wild-type luciferase just forms aggregates but this doesn't happen with various thermostable variants of it. This is what it looks like when using fusions with fluorescent proteins (Figure 5):

https://doi.org/10.17605/OSF.IO/EZU6F

Though, this preprint shows that beetle luciferases might have even more complicated behavior. 👍

Why luciferase crystallization in mammalian cells had been unrecognized despite the widespread use of firefly luciferase in numerous biomedical and life science applications? While there is a possibility that nobody took the time to report it even though this event has been known widely, there are a few potential explanations for why such a phenomenon has escaped researchers’ attention.

I must admit that I never saw any needles in HeK293 or U2OS cells, though I did work quite a lot with their adherent cultures transfected with over-expressed beetle luciferase reporters and I did actually look at cells a lot. :)

But, for example, when asking a colleague about very fast changes of cell shape of HEK293 cells in response to fresh serum, I got a response "we never look at them after changing the media". So this is certainly unappreciated feature in science. :)

There is another bioluminescence-related unappreciated feature: probably, a universal death of mammalian cells when exposed to coelenterazines in the media (e.g. after 30-60 min, as long as it's not degraded during that time, something above 20-40 uM).

To promote preprints, an interesting idea to would be to cite preprints instead of the final journal publication as long as the content, for what the citation is done, is already in the preprint. And most preprints are nearly identical to the final journal-published papers. (Essentially, it's an extension of the priority-based citation selection.)

The word "mammalian" in the title seems to misplaced by some accident: it should be e.g. "copepod" like in the abstract. Otherwise, its a really exciting family of luciferases; from practical point of view, GlucM23 and Aluc engineered mutant variants are very interesting.

When working with these, I was surprised that they just easily fold into an active form when released from E. coli, without any additional help (despite a lot of S-S bonds).

These luciferases also give a nearly complete FRET/BRET to the N-terminal mNeonGreen, so mNG-Aluc34 fusion (released from E. coli or in mammalian cells) gives essentially a pure green light.

An practically useful challenge for copepod luciferases is to make their Michaelis-Menten constants very low like decapod Nluc (Nanoluc/NanoKAZ) luciferase has (0.3–0.6 μM for typical CTZ-like substrates) because the thermodymamic solubility limits of CTZ-like substrates are quite low (5–10 μM). Though, higher substrate levels can usually be obtained as supersaturated solutions (but these can be prone to precipitation with a formation of yellow crystals: https://www.protocols.io/view/chemiluminescence-of-coelenterazine-catalyzed-by-c-5qpvoy7n9g4o/v3/materials).

A really great new substrate. It looks like by the photon output it gives even a higher activity than the native D-luciferin (if the presented activity values are not corrected for the PMT sensitivity which is usually much less sensitive to red light).