🚨 preprint 2️⃣ this month: our (purely experimental🧪) venture into #ChemBio We prouldy present: ADD-tagging of proteins (or "ADDing") —a super convenient enzymatic technique to install click chemistry handles on proteins. Led by superstar @wahyuwidodo.bsky.social www.biorxiv.org/content/10.1... A 🧵👇🏽 - ThreadSky

maxfus.bsky.social • 5 days ago

🚨 preprint 2️⃣ this month: our (purely experimental🧪) venture into #ChemBio
We prouldy present: ADD-tagging of proteins (or "ADDing") —a super convenient enzymatic technique to install click chemistry handles on proteins.
Led by superstar @wahyuwidodo.bsky.social
https://www.biorxiv.org/content/10.1101/2025.05.17.650163v1.full
A 🧵👇🏽

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brooklynkid53bskys.bsky.social•4 days ago

what are the advantages of this over say a c myc tag ?

unless the substrates are commercially avialable , and stable to typical air/water atmosphere, at a reasonable cost, this chemistry, however good it maybe , won't go far ?

maxfus.bsky.social•4 days ago

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.

andyfraser.bsky.social•5 days ago

Very neat indeed - congratulations!

carolynbertozzi.bskyverified.social•5 days ago

Congratulations on this elegant new chemoenzymatic method for site-specific protein modification 👏

maxfus.bsky.social•5 days ago

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

maxfus.bsky.social•5 days ago

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+

maxfus.bsky.social•5 days ago

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.

maxfus.bsky.social•5 days ago

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!

maxfus.bsky.social•5 days ago

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.

maxfus.bsky.social•5 days ago

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.

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