Http://www.piercenet.com/method/carbodiimide-crosslinker-chemistry

Thank you so much for such helpful ideas. I am sure I would be able to implement them.But the thing that wonder me is the iso urea byproduct. I dont want it in the mixture as i will be treating this to my cells. Is there any way of removing this byproduct from EDC made peptide-protein mixture. please input.

Dear Zaheer,

Adding a coupling agent to a protein to conjugate it to another is a very dirty way of doing things. More than the urea byproduct from the DCC you should be worried about the selectivity in the coupling: in addition to the C-terminal carboxylate, there are Asp and Glu side chains that can be activated; likewise, not only the terminal amine can react, but also Lys side chains can crosslink with the other protein. The result is a mixture of products, some of them will be active, some inactive. Not a good idea. If you know that your proteins have Cys residues, that's a far better approach to obtain conjugates in a much more selective way.

If one of your components is a peptide, then you should consider Native Chemical Ligation as a clean and selective way of reacting the two components.

Check Hermanson's book "Bioconjugate techniques" (http://amzn.to/1c9lwd9).

Is there any alternate way of joining the n and c termini of two different proteins. Like using some nucleophilic attack properties of some amino acids. If so, what could be the proper strategy that i could adopt.

Is we block the N terminal of peptide by adding some functional group and then use EDC for coupling the pepide protein, Is there any chance that self dimerization of peptide could occure as peptide also have N and C termini. Please guide in this regard. Thanks

You could synthesize the peptide with the N-terminal amine acetylated, which would make them inert towards couplings, but in order to activate the peptide acid selectively you can't have other amines (Lys) or carboxylates (Asp/Glu) in the sequence, otherwise you will have a mixture. Also, bear in mind that the target protein might react not only through its N-terminus, but also through any other Lys in the sequence. Read Hermanson's!

Dear Dr. Matar, Proteins are not regular polymers: there are many other functional groups available for reaction besides both termini. Lysines have a reactive amine, Glutamic acids and aspartates have reactive carboxylates that can compete with the C-terminal for activation and derivatization. In addition to that, the accessibility and reactivity of all those groups will be modulated by the protein structure and the particular environment created by the neighbouring residue side chains. Proteins are not like synthetic polymers. The whole field of bioconjugation evolved over the years to overcome those selectivity problems!. DCC between two proteins will NOT give a single product, or might, but in principle you can't predict—or expect—that.

Again, everything Zaheer needs to know is in Hermanson's book.

Is there any way of exploiting isopeptide strategy to make an isopeptide bond between two different protein. I attach a paper here. Please guide me on the basis of your experience if i could exploit it. It has no coupling reagent. What we need to do is to carefully design the peptide for an automatic nucleophilic attack.

Dr. Zaheer, I am told today by RG that your file is updated to include PhD. If you attained the degree recently, then congratulations.

Back to chemistry: I am attaching a 5 pages pdf file (nicely presented). I hope that it is helpful in any way.