Protein
immobilization techniques on polymeric supports have enabled
many applications in biotechnology and materials science. Attaching
the proteins with controlled orientations has inherent advantages,
but approaches for doing this have been largely limited to cysteine
or noncanonical amino acid targeting. Herein, we report a method to
attach the N-terminal positions of native proteins to polymer resins
site-specifically through the use of 2-pyridinecarboxyaldehyde (2PCA)
derivatives. For high protein loadings and practical synthesis, we
initiated this work by preparing highly reactive 2PCA derivatives
using Pd-catalyzed cross-coupling amination. The resulting compounds
were attached to amine-containing polyethylene glycol acrylamide resin
(PEGA-NH2), which subsequently reacted with the N-termini
of proteins to produce linkages that were stable over the long term
but could be reversed through the addition of hydroxylamine. We envision
that this site-selective, 2PCA-based protein immobilization can provide
a simple and generalizable immobilization protocol.
Conjugate
vaccines prepared with the cross-reactive material 197
(CRM197) carrier protein have been successful in the clinic
and are of great interest in the field of immunotherapy. One route
to preparing peptide–CRM197 conjugate vaccines involves
an activation–conjugation strategy, effectively coupling lysine
residues on the protein to cysteine thiolate groups on the peptide
of interest using a heterobifunctional linker as an activation agent.
This method has been found to result in two distinct populations of
conjugates, believed to be the result of a conformational change of
CRM197 during preparation. This report explores the factors
that lead to this conformational change, pointing to a model in which
the unintentional alkylation of histidine-21 by the activating agent
promotes the “opening” of the monomeric protein. This
exposes a new set of lysine residues that are modified by additional
activation agents. Subsequent peptide ligation to these sites results
in the two conformers. This is the first time that a specific chemical
modification is demonstrated to induce a defined conformational change
for this carrier protein. Importantly, alternative conditions and
reagents have been found to minimize this effect, improving the conformational
homogeneity of peptide–CRM197 conjugates.
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