I want to conjugate cyclic GDP to a protein to use in immunization for antibody production. Can anyone help me with a way in which I can do this conjugation?
Correction to my question. I meant cyclic-di-GMP,. Sorry for the miss writing.
You could try carbodiimide coupling of the guanine NH2 to the protein COOH.
You could also try incubating the protein with huge excess c-di-GMP at 37oC for 2-3 days and see if the protein gets glycated.
Hi Anette,
I think Steingrimur’s suggestion with carbodiimide is worth to be tried. I have no experience with the second suggestion but if you try it please report back. The problem is how can you check for proper conjugation and how can you calculate how many cyclic di-GMP molecules are crosslinked to the protein carrier. I think it is an important parameter to be taken into account before proceeding to immunization. So any input to the thread should provide some hints about that. Maybe if you have access to LC/MS facility that could solve the problem.
Thank you steingrimur for your suggestion
Is there not a risk of the NH2 or the protein reacting "faster" and easier than the NH2 of the guanin giving just crosslinking of the protein?
Hi Anette. Yes, there will definetly be a bunch of side reactions. The most prominent being protein COOH groups crosslinking with another protein NH2 groups. You can minimize those reactions by mixing together the EDC and c-di-GMP just before adding them to the protein. Also if you have, say, 5-fold molar excess of EDC and 100-fold molar excess of c-di-GMP over the protein will limit the protein crosslinking.
My second suggestion on just incubating the protein with excess nucleoside is based on the reactivity of some sugars. Some sugars (mainly aldoses) react avidly with protein NH2 goups. This reaction (Maillard) is elevated in diabetics because of the high glucose in blood.
This reaction occurs with ribose phosphate but should also occur with nucleosides. (Carbohydr Res. 2010 Nov 22;345(17):2499-506. Maillard reaction of ribose 5-phosphate generates superoxide and glycation products for bovine heart cytochrome c reduction. Gersten RA, Gretebeck LM, Hildick-Smith G, Sandwick RK).
I would be curious to see what happens if you tried that.
Good luck!
Thanks for your repsonse. I think I will go for the EDC coupling, but am also curious of how well the "plain incubation" works so I may try that as well. i will let you know how it turs out if I do.
/Anette
After consideration I decided to not try the EDC coupling
I used a lightactivated crosslinker instead, diazirine
It worked out well.
I used a NHS-ester form of diazirine which was first conjugated to KLH. After that I mixed this with c-di-GMP and exposed it to UV for 15 minutes.
Then chnaged buffer with gelfiltration, removing all non bound c-di-GMP. By analysing the absorbance spectra 220-400nm before and after conjugation and for the components alone I could verify (or at least partly verify) that the conjugation was successfull.
Another "indication of that this worked out well was that rabbits immunized with this conjugate generated antibodies specific for a c-di-GMP-BSA conjugate.
I thought I ought to let you know since you helped me out.
Thanks Anette, I'm glad it worked out for you.
Thats an interesting approach you took. Why did you choose diazirine rather than other UV activated crosslinkers?
PS
(did you try the "plain incubation")?
i conjugated cGMP to KLH or BSA with EDC and it worked. You can try to use EDC.
60-fold molar excess of cGMP over the protein I used.
good luck
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