Chad Cummings

Dramatically Increased pH and Temperature Stability of Chymotrypsin Using Dual Block Polymer-Based Protein Engineering

In this study, we report on multimodal temperature-responsive chymotrypsin-poly(sulfobetaine methacrylamide)-block-poly(N-isopropylacrylamide) (CT-pSBAm-block-pNIPAm) protein-polymer conjugates. Using polymer-based protein engineering (PBPE) with aqueous atom transfer radical polymerization (ATRP), we synthesized three different molecular weight CT-pSBAm-block-pNIPAm bioconjugates that responded structurally to both low and high temperature. In the block copolymer grown from the surface of the enzyme, upper critical solution temperature (UCST) phase transition was dependent on the chain length of the polymers in the conjugates, whereas lower critical solution temperature (LCST) phase transition was independent of molecular weight. Each CT-pSBAm-block-pNIPAm conjugate showed temperature dependent changes in substrate affinity and productivity when assayed from 0 to 40 °C. In addition, these conjugates showed higher stability to harsh conditions, including temperature, low pH, and protease degradation. Indeed, the PBPE-modified enzyme was active for over 8 h in the presence of a stomach protease at pH 1.0. Using PBPE, we created a dual zone shell surrounding each molecule of enzyme. The thickness of each zone of the shell was engineered to be separately responsive to temperature.

ATRP-grown protein-polymer conjugates containing phenylpiperazine selectively enhance transepithelial protein transport

&NA; Despite its patient‐friendliness, the oral route is not yet a viable strategy for the delivery of biomacromolecular therapeutics. This is, in part, due to the large size of proteins, which greatly limits their absorption across the intestinal epithelium. Although chemical permeation enhancers can improve macromolecular transport, their positive impact is often accompanied by toxicity. One element potentially contributing to this toxicity is the lack of co‐localization of the enhancer with the protein drug, which can result in non‐specific permeation of the intestine as well as enhancer overdosing in some areas due to non‐uniform distribution. To circumvent these issues, this study describes a new way of increasing protein permeability via a polymer conjugation process that co‐localizes permeation enhancer with the protein. Based on previous reports demonstrating the utility of 1‐phenylpiperazine as an intestinal permeation enhancer, we synthesized protein‐polymer conjugates with a phenylpiperazine‐containing polymer using polymer‐based protein engineering. A novel phenylpiperazine acrylamide monomer was synthesized and chain extended using atom transfer radical polymerization from the model protein bovine serum albumin (BSA). At non‐cytotoxic doses, the protein‐polymer conjugates induced a dose dependent reduction in the trans‐epithelial electrical resistance of Caco‐2 monolayers and an impressive ˜ 30‐fold increase in BSA permeability. Furthermore, this permeability increase was selective, as the permeability of the small molecule calcein co‐incubated with the protein‐polymer conjugate increased only 5‐fold. Together, these data represent an important first step in the development of protein polymer conjugates that facilitate selective protein transport across membranes that are typically impermeable to macromolecules. Graphical abstract Figure. No caption available.