Avidin bioconjugate with a thermoresponsive polymer for biological and pharmaceutical applications

A thermoresponsive polymer, N-isopropylacrylamide-co-acrylamide (Mn 6 kDa) with a lower critical solution temperature (LCST) of 37 degrees C, was activated and conjugated to avidin to yield a derivative with 200 kDa molecular weight. Gel permeation analysis demonstrated that the new bioconjugate possessed an apparent size corresponding to a 220 kDa globular protein. Photon correlation spectroscopy and turbidometric studies showed that the bioconjugate underwent temperature dependent phase transitions. The protein-co-polymer bioconjugate displayed the same onset phase transition temperature (LCST) as the original synthetic co-polymer. Nevertheless, the aggregation profile of the bioconjugate shifted at higher temperature as compared to the original polymer. This indicated that the aggregation behaviour coil-to-globule transition of the co-polymer was modified by anchoring to the protein surface. Circular dichroism analysis showed that the co-polymer conjugation did not alter the protein tertiary structure tertiary the aromatic amino acid environment. The bioconjugate maintained 85+/-3% of native avidin affinity for biotin and biotin-Mab, and high affinity was maintained after three heating cycles. Pharmacokinetic studies demonstrated that the co-polymer bioconjugation increased the avidin residence time in the bloodstream. The distribution phase of avidin-co-polymer was longer than the native protein by a factor of 20. The co-polymer conjugation decreased by three-fold the distribution extent of avidin and reduced significantly its up-take to the liver.