I have produced the following protein:
NH2-AGDGSG(VPGIG)25VPASGKPIPNPLLGLDSTHHHHHH-COOH
which contains a total of 3 carboxyls (2 in the side chain or Aspartate (D) and 1 C-terminus).
I have been trying to conjugate all the carboxyls (activated as NHS esters) to an amine-containing ligand, but based on the calculated conjugation efficiency it seems that only one of the 3 carboxyls is bound to the ligand.
Based on the sequence above is there any obvious reason for why 2 of the 3 carboxyls would not react?
its possible you are getting intramolecular condensation between good nucleophils like Lysine and your desired carboxyl group if they are spatially close in the folded structure
Hi Greg, I see your point but I protect the amines before conjugation. So, unless the protection is incomplete, there must be an alternative explanation.
Ok, well then i think you’d have to look at tertiary structure. I don’t think there’s anyway to tell based on primary sequence. Tertiary structure can influence reactivity. Classic example I have heard is in serine proteases there is a residue that hydrogen bonds with histidine in catalytic triad, increasing its basicity. Maybe you are having opposite effect where neighboring residues in 3D space are decreasing reactivity of your carboxyls.
Also solubility may be issue too. Are all these carboxyls on surface of protein or interior? This is again why 3D structure would help more than primary sequence.
Im just speculating though. Maybe try different activating agent like Sulfo-NHS?
Thank you Greg.
I'm about to send out a methodology paper on conjugation of amphiphilic protein to hydrophobic ligands. Would you mind if I recommend you as a reviewer? Also feel free to suggest anybody else who would find this paper interesting, and who would be willing to review it.
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