1 research outputs found
Cysteine as a Monothiol Reducing Agent to Prevent Copper-Mediated Oxidation of Interferon Beta During PEGylation by CuAAC
Bioconjugation by copper-catalyzed
azide–alkyne cycloaddition
(CuAAC) provides a powerful means to produce site-specifically modified
proteins. However, the use of a copper catalyst brings about the possible
generation of reactive oxygen species that could cause degradation
of vulnerable amino acid residues. We investigated whether PEGylation
by CuAAC caused any modifications to the therapeutic protein interferon
beta-1b, which was produced via global amino acid substitution with
azidohomo-alanine at the N-terminus and contains no methionine residues.
Using previously reported reaction conditions, LC-MS peptide mapping
detected +32 Da and +48 Da oxidation modifications of tryptic peptides
28–33 (LEYCLK) and 137–147 (EYSHCAWTIVR) in the protein
post-PEGylation. The oxidative degradation increased with reaction
time, whereas reducing the copper concentration slowed the PEGylation
rate as well as the oxidation rate. Replacing dithiothreitol (DTT)
with any of five different monothiol reducing agents in anaerobic
conditions allowed efficient PEGylation in 2–4 h and abrogated
oxidative degradation. Free cysteine provided reproducible reaction
results as a reducing agent in this system and has been successfully
applied to other protein conjugations. Monothiol reducing agents,
such as cysteine, may be useful tools as protective reducing agents
for CuAAC in some bioconjugation systems