Here is a question posed to me by one of my collegues. Any suggestions and
remarks greatly appreciated!
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"We would like to modify our protein, to introduce a single aldehyde that is
(relatively) stable. Preferably we'd like to be able to do this
reproducibly at a defined location (e.g. the N- or C- terminal). The reason
for a single modification is that we do not want to run the risk of
destroying too many protective antibody epitopes on the protein by the
addition reaction. Also, the chemistry used in the addition
reaction/stabilization has to be pretty mild - we have 6 disulfide bridges
in the protein (11 kDa) that we do not want to touch. The sort of avenue
we've been exploring is to react glyceraldehyde with our protein (addition
at primary amines). If we play with the ratios/conditions we can get to a
stoichiometry of about one addition per protein molecule. We then stabilize
the resultant Schiff base to lock the molecule in place by a mild reduction
that doesn't effect the disulfides. Subsequent treatment with sodium
periodate produces an aldehyde from the glyceraldehyde.
This approach may have limited success, but we're looking for ideas for
other approaches. For example we have thought of synthesizing a short
peptide that has an aldehyde introduced during synthesis, and then
conjugating it to the protein. However, we're not sure of the conjugation
chemistry to use to leave the aldehyde alone.
If anyone has any ideas, it would be greatly appreciated. One option of
particular appeal to me is that the protein has a C-terminal His x 6 tag.
So any attack that could produce aldehydes around those Histidines would be
ideal, as it would leave the rest of the protein alone (and provide a nice
way to separate modified from unmodified!).
PS I am not an organic chemist, so nice explanations of the chemistry
involved appreciated!"
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Jan Lukszo, Ph.D.
NIH/NIAID