maxfus.bsky.social
Asst. Prof. Uni Groningen 🇳🇱
Comp & Exp Biochemist, Protein Engineer, 'Would-be designer' (F. Arnold) | SynBio | HT Screens & Selections | Nucleic Acid Enzymes | Biocatalysis | Rstats & Datavis
https://www.fuerstlab.com
https://orcid.org/0000-0001-7720-9
325 posts
1,687 followers
730 following
Regular Contributor
Active Commenter
comment in response to
post
I'm sympathetic to your overall argument, but what do you mean with "watering down"? Surely we can train PhDs at the highest academic standards and implement more industry-readiness at the same time?
And did you mean to say "academic science"? Because the OP is about unis
comment in response to
post
deja vu
bsky.app/profile/maxf...
comment in response to
post
The backbone is fine, save for the fact that it's hallucinated
comment in response to
post
I wish someone would collect all these anecdotes / analyse systematically and compare AF3 vs its clones
comment in response to
post
bsky.app/profile/maxf...
comment in response to
post
While my uni seems to have yielded to the price hike, we don't have the full suite anymore. I ended up building our labs own interface for chemical search to replace chemfinder and if we ever abandon chemdraw I quite like
app.molview.com
comment in response to
post
Looks like a fun challenge! Did u implement a simple way to run new approaches with the benchmark? I.e. if I have a script that takes imgs as input, how does output have to be formatted & how do I run the benchmark on the outcome?
My first idea would be img segmentation, have you tried already?
comment in response to
post
I'm as skeptical as anyone bout chem synth with AI, but the quote
-actually finds model got synthesis right (appar tricky as OP messed up in lab & prob. wasted days-weeks)
-appar misprompted multiple times
-finds new visual feature still buggy (how DARE they!)
-ends with plug for paywalled article
😶🌫️
comment in response to
post
The substrates (NAD+ and the alkanol) are commercially available, even the substrate generating enzyme is available at sigma, and a reaction costs <1$
The bond is stable under all conditions we tested, up to 100⁰C, pH 2-10, and typical phosphatases didn't remove it either.
comment in response to
post
Congratulations to Wahyu and thanks for his very hard work!
Feedback highly welcome, link to preprint again:
www.biorxiv.org/content/10.1...
comment in response to
post
3) Perhaps most importantly for pharma (e.g. antibody drug conjugates): the molecular scar connecting protein to functional group is minimal and manifests as a phosphodiester bond - ubiquitously found in nature (→low immunogenicity?!)
comment in response to
post
1) ApbE is easily expressed in E. coli, only requires Mg2+, and its recognition motif DxxxGA[T/S] is short, flexible, and works at N and Cterm + internally.
2) Donor substrates can be made w/o chemical synthesis via a commercially avail. enzyme & if desired in the same reaction as bioconjugation
comment in response to
post
In summary, ADDing is a very convenient new way to install click-chemistry handles to your favorite protein for subsequent bioconjugation. We'd be hapy to share reagents for anyone wanting to try! Compared to other systems (sortase, transglutaminase, Sfp, etc), we see many advantages:
comment in response to
post
As a final application example, we demonstrate protein immobiliziation. Commercial azido agarose reacted happily with ADD-tagged GFP, and we got a brightly shining resin after reacting the protein on-column with ApbE!
comment in response to
post
Conjugating commercial click-modified DNA oligos also worked at the first shot without optimization, although at relatively low conjugation yields. We’re not so experienced with the chemistry, but apparently Cu catalysis isn’t great for DNA either, so probably switching to SPAAC would work better.
comment in response to
post
As expected, biomolecule conjugation needs more care: we saw moderate conversion yields in protein-protein conjugations (N-N/C-C/branched). We did not optimize our proof of concept—safe to assume that it could easily be pushed by varying position, adding linkers, optimized reaction conditions, etc.
comment in response to
post
Small molecules were readily conjugated to all our example proteins: we show attachment of fluorophores & biotin, and establish that both classic copper-catalyzed, as well as @carolynbertozzi.bskyverified.social's copper-free SPAAC reaction are compatible with ADD tags & result in full labeling.
comment in response to
post
As labeling was so straightforward, we dedicated our remaining efforts on demonstrating ADDing’s compatibility with common use cases: bioconjugation of small molecules, biomolecules, and protein immobiliziation.
comment in response to
post
Because ADPRC works on NAD+, not NADPH, while ApbE only accepts NADPH and ignores NAD+, we can even run the two enzymatic steps (substrate generation & protein modification) in one pot—just add the two enzymes with the precursors NAD+ and the click alkanol to your target protein!
comment in response to
post
Turns out ApbE is happily accepting this substrate, allowing us to label a variety of proteins at N or C terminus, as well as internally, with either alkynyl or azido ribose phosphate moieties, yielding typically full conversion after a few hours of reaction, as determined by MS.
comment in response to
post
Given the enzyme’s reported promiscuity toward NADH, we hypothesised that it tolerates synthetic substrates with similar structure. Courtesy to Jäschke lab developing NAD capture seq, we knew that a commercial enzyme—ADPRC—can generate a click dinucleotide from cheap small molecule alcohols & NAD+
comment in response to
post
For reasons that will hopefully become clear in future, our interdisciplinary lab has been looking into protein modifications for a while.
My colleague Marco @fraaije.bsky.social recently described the Flavin tag, a great way to attach FMN on a short peptide tag, using the flavin transferase ApbE
comment in response to
post
thanks for sharing, we should try these! Will think if there's a way to get on protein, but if not, I guess they could be on the small molecule partner instead
comment in response to
post
thanks so much for the mini review!!
comment in response to
post
I'd be v. sure that the motif is specific enough to not occur by chance on some other important protein in ur sample. Currently not yet thinking in vivo where it might be an issue in some cases
we did compare, but so many systems & considerations - takes a whole review to explain why ours is best :)
comment in response to
post
Best option is probably case-specific. Let's say ur target has His-tag, u could produce ApbE with another tag, say strep, and get ur modified protein back with Ni-NTA, or even do on-column mod). Separating mod vs non-mod would be trickier for small mol mods than biomols, but there we see 100% yield
comment in response to
post
I have to admit I was not aware of them until now, so - no idea. will have a look, thanks!
comment in response to
post
"there's two types of drivers - the idiots that go slower than me, and the maniacs who go faster than me"
Is the equivalent length of manuscript or number of replicates 🤔
comment in response to
post
Plenty of biomolecules, the only synthetic reaction is click chemistry, and all off the shelf reagents
comment in response to
post
So glad you asked, and I'll of course recommend our own system hot off the press
www.biorxiv.org/content/10.1...
Bluetorial coming later today :)
comment in response to
post
Ain't got no numbers, but click conjugates must be close in wide availability, including DNA. My point is, using enz labeling goves you plenty of advantages on top of overcoming the issues you mentioned in op