The attachment of inert polymers, such as polyethylene glycol (PEG), to proteins has driven the emergence of a multibillion dollar biotechnology industry. In all cases, proteins have been stabilized or altered by covalently coupling the pre-existing polymer to the surface of the protein. This approach is inherently limited by a lack of exquisite control of polymer architecture, site and density of attachment. Polymer-based protein engineering (PBPE) with a novel water-soluble atom transfer radical polymerization (ATRP) initiator was used to design temperature responsive enzyme-polymer conjugates using the model enzyme chymotrypsin. By using a “grafting from” approach, we synthesized homogenous enzyme-polymer conjugates by first covalently coupling the ATRP initiator to over 85% of accessible surface lysines followed by polymerization outward from the enzyme surface. The growth of a dual temperature-responsive block copolymer, poly(sulfobetaine methacrylamide)-block-poly(N-isopropyl acrylamide) (pSBAM-b-pNIPAm) from the surface of chymotrypsin made it possible to predictably alter chymotrypsin-polymer conjugate activity, productivity, stability, substrate affinity, and structural conformation. Specifically, chymotrypsin-pSBAm-block-pNIPAm conjugates showed temperature dependent changes in substrate affinity and productivity when cycled above or below their critical solution temperatures. Most interestingly, the chymotrypsin conjugates showed dramatically increased stability in low pH conditions when compared to native chymotrypsin. Further work with chymotrypsin revealed that a similarly structured zwitterionic polymer, poly(carboxybetaine acrylamide) (pCBAM) also stabilized chymotrypsin to low pH. Separate chymotrypsin conjugates synthesized with pCBAM or poly(oligo ethylene glycol methacrylate) (pOEGMA) were investigated with further experiments to reveal CT structural differences dependent on the attached polymer.

Densely modified chymotrypsin-polymer conjugates with increased stability to low pH

251st National Meeting (2016)

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