3 research outputs found
Protein–Polymer Conjugation via Ligand Affinity and Photoactivation of Glutathione <i>S</i>‑Transferase
A photoactivated,
site-selective conjugation of polyÂ(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
Correction to Trehalose Glycopolymers as Excipients for Protein Stabilization
Correction to Trehalose Glycopolymers as Excipients
for Protein Stabilizatio
Trehalose Glycopolymers as Excipients for Protein Stabilization
Herein,
the synthesis of four different trehalose glycopolymers
and investigation of their ability to stabilize proteins to heat and
lyophilization stress are described. The disaccharide, α,α-trehalose,
was modified with a styrenyl acetal, methacrylate acetal, styrenyl
ether, or methacrylate moiety resulting in four different monomers.
These monomers were then separately polymerized using free radical
polymerization with azobisisobutyronitrile (AIBN) as an initiator
to synthesize the glycopolymers. Horseradish peroxidase and glucose
oxidase were incubated at 70 and 50 °C, respectively, and β-galactosidase
was lyophilized multiple times in the presence of various ratios of
the polymers or trehalose. The protein activities were subsequently
tested and found to be significantly higher when the polymers were
present during the stress compared to no additive and to equivalent
amounts of trehalose. Different molecular weights (10 kDa, 20 kDa,
and 40 kDa) were tested, and all were equivalent in their stabilization
ability. However, some subtle differences were observed regarding
stabilization ability between the different polymer samples, depending
on the stress. Small molecules such as benzyl ether trehalose were
not better stabilizers than trehalose, and the trehalose monomer decreased
protein activity, suggesting that hydrophobized trehalose was not
sufficient and that the polymeric structure was required. In addition,
cytotoxicity studies with NIH 3T3 mouse embryonic fibroblast cells,
RAW 264.7 murine macrophages, human dermal fibroblasts (HDFs), and
human umbilical vein endothelial cells (HUVECs) were conducted with
polymer concentrations up to 8 mg/mL. The data showed that all four
polymers were noncytotoxic for all tested concentrations. The results
together suggest that trehalose glycopolymers are promising as additives
to protect proteins from a variety of stressors