This review summarizes our recent efforts on protein stabilization by polymer conjugation. The first part focuses on molecular dynamics simulation of PEGlyated insulin, in which an annealing procedure was proposed that enabled an all-atom level examination of the interaction between PEG polymers of different chain lengths and insulin. It was shown that PEG entangled around protein surface through hydrophobic interaction and concurrently formed hydrogen bonds with the surrounding water molecules. In addition to an enhanced structural stability, conjugation gave an increased size while a decreased the solvent accessible surface area. These favored a prolonged circulation life despite kidney filtration, proteolysis, and immunogenic side effects. The simulation that reproduced the experimental results in literates provided a molecular insight into the PEGlayted protein.
The second part presents a novel procedure to fabricate uniform protein-polymer conjugate with improved stability under adverse conditions. This method was based on in situ aqueous activators generated by electron transfer for atom transfer radical polymerization (AGET-ATRP) in air, using enzyme as macroinitiator. The procedure started by grafting 2-bromoisobutyryl bromide (BIB) as initiator onto the protein surface, followed by in situ AGET ATRP polymerization in air using CuBr2/1,1,4,7,7-pentamethyldiethylenetriamine (PMDTA) as a catalyst and ascorbic acid as a reducing agent. The size of conjugate was shown to be a function of monomer loading and reaction time. The effectiveness of this method has been demonstrated using horseradish peroxidase (HRP) as a model protein and acrylamide as the monomer. The yielded HRP conjugates essentially reproduced the catalytic behavior of HRP in free form but exhibited significantly enhanced thermal stability against high temperature and trypsin digestion. One noteworthy advantage of this procedure that it prevented the formation of copolymer and thus facilitated purification of the protein conjugate.
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