Excellent dive by Sasha using a Glock analogy I can follow! -JP
Does Famotidine Directly Bind and Act as an Inhibitor of SARS-CoV-2 PLpro?
Production of Recombinant SARS-CoV-2 Plpro
An expression plasmid containing the sequence for (His)6-TEVsite-SARS-CoV-2 PLpro (nsp3 from Wuhan-Hu-1 isolate, polyprotein 1ab 1564-1878) was obtained commercially from ATUM. The plasmid was transformed into E. coli BL21 (DE3) pLysS. The expression and purification protocols were adapted from prior work (Lindner et al., 2005).

Production of Recombinant ISG15
The expression plasmid for proISG15 (2-165) was a gift from David Komander (Addgene plasmid #110762; http://n2t.net/addgene:110762 ; RRID:Addgene_110762) (Swatek et al., 2018). Expression and purification protocols were adapted from (Swatek et al., 2018). A size exclusion chromatography step on a Superdex 75 column (GE Healthcare) was added as a final step.

PLpro Activity Assays
Cleavage of ISG15 by SARS-CoV-2 PLpro was tested by incubating 4 nM of PLpro in 50 mM Tris-HCl (pH 7.3), 150 mM NaCl, 2 mM DTT, 0.1 mg ml−1 BSA, with 10 µM of ISG15 in a final volume of 20 µL for 1 h at room temperature. Control was incubated without enzyme. Samples were subjected to SDS-PAGE.

First - what is SARS-CoV-2 papain-like protease (PLpro)?

A protease is an enzyme that catalyzes proteolysis, breaking down proteins into smaller polypeptides or single amino acids, and spurring the formation of new protein products.

What do they mean by “papain-like”?

Turns out, they mean what it sounds like - papaya. As in fruit. As in … remember the assassinated President of Tanzania who tested papaya for covid and got a positive? Yes, about that: Papain, also known as papaya proteinase I, is a cysteine protease (EC 3.4.22.2) enzyme present in papaya (Carica papaya) and mountain papaya (Vasconcellea cundinamarcensis). It is the namesake member of the papain-like protease family. It has wide ranging commercial applications in the leather, cosmetic, textiles, detergents, food and pharmaceutical industries. In the food industry, papain is used as an active ingredient in many commercial meat tenderizers.

Ok, so this super novel deadly pandemic virus that may or may not have jumped form a bat, or maybe was engineered in Wuhan (damn you, CCP Chynaaaa!!) seems to have common parts with papaya, and also a goat and Coca-Cola and commercially available detergents… The papaya enzyme is a common chemical in pharmaceutical manufacture of synthetic products… M-kay…

So, this is where Bob used “real live virus” like he adamantly claims in the video interview, right?

Wrong.

The research team DID NOT use anything resembling the “real live virus” in this part of the experiment. What they used instead was a plasmid obtained from a commercial manufacturer (ATUM) which design is based on a small part of the allegedly “sequenced genome” of the alleged Wuhan-1 variant. The same piece just happens to be widely commercially used in all sorts of chemical manufacture, so that’s convenient!

Maybe he didn’t read his own paper? Idk, he is a busy man…

Does Famotidine Directly Bind and Act as an Inhibitor of SARS-CoV-2 PLpro? Production of Recombinant SARS-CoV-2 Plpro. An expression plasmid containing the sequence for (His)6- TEVsite-SARS-CoV-2 PLpro (nsp3 from Wuhan-Hu-1 isolate, polyprotein 1ab 1564-1878) was obtained commercially from ATUM. The plasmid was transformed into E. coli BL21 (DE3) pLysS. The expression and purification protocols were adapted from prior work (Lindner et al., 2005).

The plasmid was designed to produce (express) the LPro papaya thingy when added to a brew of chemicals (so molecular biology always claims). But we already established that the papaya thingy LPro is not a virus, but only claimed to be part of it by those people who uploaded the “covid sequence” into GenBank, based on 1 Chinese patient nobody ever saw or has a record of…. And neither it is “live”… It’s a synthetic chemical molecule, and only because of its very large size its is called a biologic product as opposed to a regular small-molecule chemical. You see, synthetic leather covering your chair is boring, but synthetic biology is exiting!

But wait, they used another thing, which Bob angrily claims was given to them by a big name researcher - ISG15! Is that a “live virus”?

No.

ISG15 (Interferon-Stimulated Gene 15) protein is minimally expressed under normal physiological conditions. However, it is aberrantly elevated in various human diseases, including multiple types of cancer, neurodegenerative disorders, inflammatory diseases, bacterial and viral infections, traumatic brain injury and many others. ISG15 is now considered diagnostic/prognostic biomarker and a therapeutic target for these ailments.

That ISG15 thing is a “target”. For example, if you go to the range to test your Glock, you can bring a paper target. ISG15 in this case played the role the target to set up an assay - an investigative (analytic) procedure for qualitatively or quantitatively measuring the presence, amount, or functional activity of a target entity. This assay would be measuring if there is a lot, a little or no papaya-thingy activity in various concoctions of tested drugs.

PLpro Activity Assays

Cleavage of ISG15 by SARS-CoV-2 PLpro was tested by incubating 4 nM of PLpro in 50 mM Tris-HCl (pH 7.3), 150 mM NaCl, 2 mM DTT, 0.1 mg ml−1 BSA, with 10 µM of ISG15 in a final volume of 20 µL for 1 h at room temperature. Control was incubated without enzyme. Samples were subjected to SDS-PAGE.

However, ISG15 is the broadest possible target, activated by almost any human illness and injury. In my firing range analogy, the target would be the size of your entire range from one end to another. So, if you hit it, you ain’t a marksman.

And in this paper nobody bothered to describe how they validated that ATUM shipped the correct papaya thingy to Bob’s team! You see, when papaya-thingy plasmid comes from ATUM or another shop like them all you get is an empty vial. And a piece of paper saying that there is a magic spell in the vial based on GenBank or some other place, and for an extra $9.95 they named a star after you. They could drop a bit of papaya and some goat urine and/or Coca-Cola into that vial instead and, chances are, the assay with ISG15 will work, because it’s activated by just about anything. By the way, that’s how all the covid tests “work” if you ever wondered.
https://sashalatypova.substack.com/p/famoti-gate?utm_source=post-email-title&publication_id=870364&post_id=141058278&utm_campaign=email-post-title&isFreemail=true&r=mvqcr&open=false&utm_medium=email