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You know if we are desperate enough it would be quite easy to make your own purified recombinant covid spike protein in bacteria. This could be used for a vaccine without too much regulatory. Protein could be made in a few weeks. Need to be sterile of course, dosage worked out. I've only immunized rabbits with these types of preps. Always get good antibodies,no harm to rabbits.
djwr...@gmail.com
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Apr 5, 2020, 8:38:25 PM4/5/20
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I think we are desperate enough. Let’s do it. Step one. Do we need the entire spike protein or just a portion for antibodies?
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> On Apr 5, 2020, at 4:02 PM, Zonie deep <deck...@gmail.com> wrote:
>
> You know if we are desperate enough it would be quite easy to make your own purified recombinant covid spike protein in bacteria. This could be used for a vaccine without too much regulatory. Protein could be made in a few weeks. Need to be sterile of course, dosage worked out. I've only immunized rabbits with these types of preps. Always get good antibodies,no harm to rabbits.
>
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Bryan Jones
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Apr 5, 2020, 10:25:41 PM4/5/20
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You might get a different response if you use a portion than if you used whole protein. Specifically, you are probably less likely to produce neutralizing antibodies from a short peptide. Ideally, you'd probably want to leave off the membrane anchor part of the protein. Maybe a soluble construct like the one used here: http://dx.doi.org/10.1016/j.cell.2020.02.058 It might be important to get human-like glycosylation, so E. coli or Sacromyces might not be the best expression system.
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I think at this stage, the input that computational modeling could provide may show if any part of the sequence to be used can sufficiently bind the virus. If binding does occur computationally, then it might be worth it to proceed with testing such sequence.
MERS-CoV genome is assembled similar to other CoVs, and the gene coding for S is located in the one-third of the genome at the 3′ end (Fig. 2A). S is a type I, trimeric, transmembrane protein located at the surface of the viral envelope, giving rise to spike-shaped protrusions from the virion. S is 1353 amino acids in length, heavily glycosylated (with 21 predicted N-linked glycosylation sites), and consists of a large ectodomain and a short cytosolic tail (Fig. 2B). The S proteins of CoVs can be divided into two functional subunits (Xia et al., 2014): the N-terminal S1 subunit forms the globular head, and the membrane-embedded C-terminal S2 region forms the stalk region. S1 is involved in receptor recognition and binding and is less conserved compared to the stalk. Two discrete, independently folded domains are located at the N- and C-termini of S1 and are able to bind receptors.
Zonie deep
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Apr 6, 2020, 1:13:19 AM4/6/20
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I would say about 150 amino acids from the receptor binding domain of the spike protein. These guys mapped optimal regions of the spike protein to make neutralizing antibodies against.
Just need spike protein cdna to pcr off or have the DNA synthesized. Then clone into bacteria expression vector like pgex or pet. Can purify with glutathione or talon beads.
Zonie deep
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Apr 6, 2020, 1:13:19 AM4/6/20
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Hi from the modeling or crystal structures I've seen S1 subunit binding to cell surface receptor(s) involves amino acids in the receptor binding region of S1. No evidence that glycosylated regions involved in binding.
This would be good since a lot easier to make protein in E. coli especially when resources are limited.
Zonie deep
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Apr 6, 2020, 1:26:55 AM4/6/20
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Ok....not sure about all this since I believe the devil is always in the details, but I have no expertise here and won't pass judgment.
My only comment is once you have your cDNA, make sure you alter the sequence to prioritize synonymous codons favored for expression in E. Coli or whatever you are inserting this into. I don't know how far of codon usage bias or even the genetic code is between SARS-CoV-2 and E. Coli but I suspect there are important differences.
Zonie deep
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Apr 7, 2020, 11:20:34 AM4/7/20
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Never had a problem with codon usage. Bacteriophage love E. coli. Many large proteins degraded when expressed in bacteria but small pieces like this usually quite stable and highly expressed. Especially when made as fusion proteins. To express like this is routine. Many companies have made spike protein already.
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Related to glycosylation differences in humans versus other species in which to raise antibodies I recently read an article about the Canadian Quebec company Medicago having patents for glysolyation pattern modifications in the host plants and ramping up production of massive quantities of COVID-19 antigen to be used for vaccinations with government support. See this link: https://www.medicago.com/en/newsroom/
Ironically the host plant species is tobacco and not medicago which is the company name. medicago is a legume genus that includes the species alpha alpha.
Anyways my question was whether anyone here is familiar with what glysolylation modifications medicago has been awarded patent protection for. I am traveling with spotty internet connectivity, or I would get on a desktop and check CAMBIA's lens or WIPO's patentscope. can anyone help
This email was text to speech so please pardon complete lack of punctuation. Thank you. Stay safe.
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Hi Zonie deep (and everyone else),
Could you post a protocol of what you intend to do, including any and all materials that would need to be bought?
Mike
On Tuesday, April 7, 2020 at 1:58:26 PM UTC-7, Tom De Medts wrote:
Related to glycosylation differences in humans versus other species in which to raise antibodies I recently read an article about the Canadian Quebec company Medicago having patents for glysolyation pattern modifications in the host plants and ramping up production of massive quantities of COVID-19 antigen to be used for vaccinations with government support. See this link: https://www.medicago.com/en/newsroom/
Ironically the host plant species is tobacco and not medicago which is the company name. medicago is a legume genus that includes the species alpha alpha.
Anyways my question was whether anyone here is familiar with what glysolylation modifications medicago has been awarded patent protection for. I am traveling with spotty internet connectivity, or I would get on a desktop and check CAMBIA's lens or WIPO's patentscope. can anyone help
This email was text to speech so please pardon complete lack of punctuation. Thank you. Stay safe.
On Tue, Apr 7, 2020 at 11:20 AM Zonie deep <deck...@gmail.com> wrote:
Never had a problem with codon usage. Bacteriophage love E. coli. Many large proteins degraded when expressed in bacteria but small pieces like this usually quite stable and highly expressed. Especially when made as fusion proteins. To express like this is routine. Many companies have made spike protein already.
Ok....not sure about all this since I believe the devil is always in the details, but I have no expertise here and won't pass judgment.
My only comment is once you have your cDNA, make sure you alter the sequence to prioritize synonymous codons favored for expression in E. Coli or whatever you are inserting this into. I don't know how far of codon usage bias or even the genetic code is between SARS-CoV-2 and E. Coli but I suspect there are important differences.
On Sunday, April 5, 2020 at 7:02:06 PM UTC-4, Zonie deep wrote:
You know if we are desperate enough it would be quite easy to make your own purified recombinant covid spike protein in bacteria. This could be used for a vaccine without too much regulatory. Protein could be made in a few weeks. Need to be sterile of course, dosage worked out. I've only immunized rabbits with these types of preps. Always get good antibodies,no harm to rabbits.
