Hello all,
I am interested in chemically modifying an expressed and purified protein by carbamylating its N-terminus. The difficulty is that most protocols I've found in the literature use 8 M urea to denature the protein so that lysine and to some degree arginine residues will also be carbamylated. Most of these are mass spec studies, so it's irrelevant that the protein gets denatured in the process.
I am interested in studying the native state of my protein, so the use of high denaturant concentrations for carbamylation isn't optimal. I guess I could refold after the carbamylation reaction, but refolding often brings its own set of problems.
My question: does anybody know of a protocol for N-terminally carbamylating proteins that uses isocyanate directly (instead of depending on urea to generate the isocyanate), or does anybody know of some other protocol altogether? Thanks in advance.
Hi Matthew, CNBr, as in the activated chromatography resin, generates carbamate (isourea) bonds with primary amines at physiological conditions.
The problem is that, aside from minor pKa differences, all primary amines can be modified with this and other amine modifying reagents.
Hello Matthew,
I agree with Steingrimur that all primary amines will be modified.
Therefore, it would be interesting to know how many lysines you have. If you do not have any or a few, you can mutate these residues into alanine or arginine unless the lysine residues do not play a role in folding, activation of protein etc.
If it is not the case, you can synthesize small peptides derived from your protein and try to carbamylate these on the N-termini with CNBr.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques