High Density Polymer Modification of Proteins Using Polymer - Based Protein Engineering

<p>Proteins and protein-based materials are used for a wide range of therapeutic, diagnostic, and biotechnological applications. Still, the inherent instability of proteins in non-native environments greatly limits the applications in which they are effective. In order to increase their utility, proteins are often modified, either biologically or chemically, to manipulate their bioactivity and stability profiles. In this work, covalent attachment of polymers to the enzyme chymotrypsin was used to predictably tailor protein bioactivity and stability. Specifically, atom transfer radical polymerization (ATRP) based polymer-based protein engineering (PBPE) was used to grow polymers directly from the surface of chymotrypsin. First, the temperature responsive polymers poly(N-isopropyl acrylamide) (pNIPAM), which has a lower critical solution temperature (LCST) and poly(dimethylamino propane sulfonate) (pDMAPS), which has an upper critical solution temperature (UCST), were separately grown from chymotrypsin. The temperature responsive properties of the polymers were conserved in the protein-polymer conjugates, and chymotrypsin bioactivity, productivity, and substrate specificity were predictably tailored at different temperatures depending on the structural organization of the polymers. Next, a dual block polymer-chymotrypsin conjugate was synthesized by growing poly(sulfobetaine methacrylamide) (pSBAm)-block-pNIPAm conjugates from the surface of chymotrypsin. The CT-pSBAm-b-pNIPAm conjugates showed temperature dependent kinetics, due to UCST or LCST driven polymer collapse at high and low temperature. Most interestingly, the dual block conjugates were dramatically more stable than native chymotrypsin to low pH. In order to further investigate the effect of polymer conjugation on chymotrypsin stability at low pH, four distinct and uniquely charged polymers were grown from the surface of chymotrypsin. With these new conjugates, we confirmed that chymotrypsin low pH stability was dependent on the chemical structure of polymers covalently attached to chymotrypsin. Indeed, positively charged polymers stabilized chymotrypsin to low pH, but negatively charged and amphiphilic polymers destabilized the enzyme. Lastly, after developing strategies for low pH stabilization, new protein-polymer conjugates with the chemical permeation enhancer 1-phenylpiperazine were designed to enable protein transport across the intestinal epithelium. Bovine serum albumin-poly(oligoethylene methacrylate)-block-poly(phenylpiperazine acrylamide) BSA-pOEGMA-b-pPPZ conjugates induced dose dependent increases in Caco-2 monolayer permeability and transported across an in vitro intestinal monolayer model with low cell toxicity.