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LPS conjugate

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Barlevy

Participant

Posts: 3

Joined: Sun Nov 29, 2020 12:00 pm

Post Tue Dec 01, 2020 6:25 am

LPS conjugate

I want to conjugate LPS to x8 polymer. Is there any simple way? I found that there is a LPS-FITC conjugate, is there anything similar either to buy or to prepare?
Also, what is the structure of x8?
Thank you in advance!
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mleipold

Guru

Posts: 5792

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Tue Dec 01, 2020 4:10 pm

Re: LPS conjugate

Hi Bar,

What is your overall goal/plan for the LPS conjugation?

The reason I ask, this isn't a straightforward question. For one, "LPS" isn't just one thing: it varies greatly among Gram-negative species. Even within the same species (eg, E. coli), different strains can have great differences in the Lipid A (lipid anchors+Kdo), inner core (sugars just after the Lipid A), outer core (sugars just after the inner core), and O-antigen sections (including the inherent presence/smooth strain vs absent/rough strain aspect). Even within the same strain, things can change depending on growth conditions.

So, what might work for conjugation in one case might *not* work in another case.


The actual structure of X8 has not been formally declared by Fluidigm. However, the references of various data sheets and some talks given over the years basically imply that X8 is a shorter version of the linear polymer described here ( http://dx.doi.org/10.1002/anie.200700796 ), but with DTPA rather than DOTA as chelating groups.


Mike
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Barlevy

Participant

Posts: 3

Joined: Sun Nov 29, 2020 12:00 pm

Post Sun Dec 06, 2020 12:37 pm

Re: LPS conjugate

Hi Mike,
Thanks for your reply. Our plan is to evaluate binding of LPS in the presence of inhibitors. These varieties do change things a little and we need to make sure we use the same LPS we're currently using. We are looking for a prep similar to:
https://www.sigmaaldrich.com/catalog/pr ... &region=IL

Thank you for X8 information.
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mleipold

Guru

Posts: 5792

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Mon Dec 07, 2020 4:28 pm

Re: LPS conjugate

Hi Bar,

That LPS link from Sigma says it's E. coli O111:B4.

That appears to be an R2 outer core strain ( https://www.ncbi.nlm.nih.gov/pmc/articles/PMC97256/ ) and has an overall LPS structure shown in Fig 2 here: https://www.researchgate.net/publicatio ... s_A_review

My assumption would be that the FITC(s) in the Sigma link would be attached at the primary amines just above the "Cell Exterior" line in the figure. I guess it's also possible that some of the GlcNAc and GalNAc in the O-antigen might not be fully acetylated, leaving some other amines to conjugate to.


To attach X8 to this, you'd have to reverse engineer some things like I did in the protocol in this link, to conjugate X8/DN3 to streptavidin: viewtopic.php?f=7&t=1016&p=3022&hilit=streptavidin#p3022 (see Apr 12 2018 post)


Mike

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