AOT (120 mg) and Brij 30 (230 mg) were added to a glass vial (20 ml) to which a stir bar was added. The vial was sealed with a Teflon-lined septum cap and purged with dry nitrogen for 10 min. Nitrogen-deoxygenated hexane (5 ml) was then added to the vial under vigorous stirring. For the aqueous phase, uricase (premodified with acryloyl group, 1 mg) was dissolved in Hepes buffer (125 μl, pH 8.5) to which TMAO monomer (50 mg) was added and dissolved. Dry nitrogen was bubbled through the monomer/protein solution for 2 min, after which the aqueous phase was slowly added to the organic continuous phase dropwise. The vial was sonicated to form a stable microemulsion. A 20% (w/v) solution of APS (10 μl) in Milli-Q water was then added to the emulsion. After 5 min, polymerization was initiated by the addition of TEMED (6 μl) and maintained at 4°C under rapid magnetic stirring. After the 2-hour reaction, the organic solvent was removed by rotary evaporator, and the PTMAO-uricase conjugate was precipitated and washed with THF three times. The PTMAO-uricase conjugate was resuspended in PBS buffer and purified with high-resolution SEC (Sephacryl S-500HR) to remove the free uricase. Lastly, the conjugates were washed and concentrated with PBS (pH 7.4) three times using a 100-kDa molecular mass cutoff centrifugal filter. The protein-polymer conjugates were characterized by GPC (Wyatt technology).

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