Protein–Polymer Conjugation via Ligand Affinity
and Photoactivation of Glutathione <i>S</i>‑Transferase
- Publication date
- Publisher
Abstract
A photoactivated,
site-selective conjugation of poly(ethylene glycol)
(PEG) to the glutathione (GSH) binding pocket of glutathione <i>S</i>-transferase (GST) is described. To achieve this, a GSH
analogue (GSH-BP) was designed and chemically synthesized with three
functionalities: (1) the binding affinity of GSH to GST, (2) a free
thiol for polymer functionalization, and (3) a photoreactive benzophenone
(BP) component. Different molecular weights (2 kDa, 5 kDa, and 20
kDa) of GSH-BP modified PEGs (GSBP-PEGs) were synthesized and showed
conjugation efficiencies between 52% and 76% to GST. Diazirine (DA)
PEG were also prepared but gave conjugation yields lower than for
GSBP-PEGs. PEGs with different end-groups were also synthesized to
validate the importance of each component in the end-group design.
End-groups included glutathione (GS-PEG) and benzophenone (BP-PEG).
Results showed that both GSH and BP were crucial for successful conjugation
to GST. In addition, conjugations of 5 kDa GSBP-PEG to different proteins
were investigated, including bovine serum albumin (BSA), lysozyme
(Lyz), ubiquitin (Ubq), and GST-fused ubiquitin (GST-Ubq) to ensure
specific binding to GST. By combining noncovalent and covalent interactions,
we have developed a new phototriggered protein–polymer conjugation
method that is generally applicable to GST-fusion proteins