title: Design of Stomach Acid-Stable and Mucin-Binding Enzyme Polymer Conjugates.
abstract: The reduced immunogenicity and increased stability of protein-polymer conjugates has made their use in therapeutic applications particularly attractive. However, the physicochemical interactions between polymer and protein, as well as the effect of this interaction on protein activity and stability, are still not fully understood. In this work, polymer-based protein engineering was used to examine the role of polymer physicochemical properties on the activity and stability of the chymotrypsin-polymer conjugates and their degree of binding to intestinal mucin. Four different chymotrypsin-polymer conjugates, each with the same polymer density, were synthesized using "grafting-from" atom transfer radical polymerization. The influence of polymer charge on chymotrypsin-polymer conjugate mucin binding, bioactivity, and stability in stomach acid was determined. Cationic polymers covalently attached to chymotrypsin showed high mucin binding, while zwitterionic, uncharged, and anionic polymers showed no mucin binding. Cationic polymers also increased chymotrypsin activity from pH 6-8, while zwitterionic polymers had no effect, and uncharged and anionic polymers decreased enzyme activity. Lastly, cationic polymers decreased the tendency of chymotrypsin to structurally unfold at extremely low pH, while uncharged and anionic polymers induced unfolding more quickly. We hypothesized that when polymers are covalently attached to the surface of a protein, the degree to which those polymers interact with the protein surface is the predominant determinant of whether the polymer will stabilize or inactivate the protein. Preferential interactions between the polymer and the protein lead to removal of water from the surface of the protein, and this, we believe, inactivates the enzyme.
authors.affiliation: Center for Polymer-Based Protein Engineering, ‡Department of Biomedical Engineering, §Disruptive Health Technology Institute, and ∥Department of Chemistry, Carnegie Mellon University , Pittsburgh, Pennsylvania 15213, United States.
authors.forename: Chad S
authors.initials: CS
authors.lastname: Cummings
authors.affiliation: Center for Polymer-Based Protein Engineering, ‡Department of Biomedical Engineering, §Disruptive Health Technology Institute, and ∥Department of Chemistry, Carnegie Mellon University , Pittsburgh, Pennsylvania 15213, United States.
authors.forename: Alan S
authors.initials: AS
authors.lastname: Campbell
authors.affiliation: Center for Polymer-Based Protein Engineering, ‡Department of Biomedical Engineering, §Disruptive Health Technology Institute, and ∥Department of Chemistry, Carnegie Mellon University , Pittsburgh, Pennsylvania 15213, United States.
authors.forename: Stefanie L
authors.initials: SL
authors.lastname: Baker
authors.affiliation: Center for Polymer-Based Protein Engineering, ‡Department of Biomedical Engineering, §Disruptive Health Technology Institute, and ∥Department of Chemistry, Carnegie Mellon University , Pittsburgh, Pennsylvania 15213, United States.
authors.forename: Sheiliza
authors.initials: S
authors.lastname: Carmali
authors.affiliation: Center for Polymer-Based Protein Engineering, ‡Department of Biomedical Engineering, §Disruptive Health Technology Institute, and ∥Department of Chemistry, Carnegie Mellon University , Pittsburgh, Pennsylvania 15213, United States.
authors.forename: Hironobu
authors.initials: H
authors.lastname: Murata
authors.affiliation: Center for Polymer-Based Protein Engineering, ‡Department of Biomedical Engineering, §Disruptive Health Technology Institute, and ∥Department of Chemistry, Carnegie Mellon University , Pittsburgh, Pennsylvania 15213, United States.
authors.forename: Alan J
authors.identifier: 0000-0001-5101-4371
authors.initials: AJ
authors.lastname: Russell
mesh_terms: D002448:Cell Adhesion; D002918:Chymotrypsin / Q000737:chemistry / Q000378:metabolism*; D005744:Gastric Acid / Q000737:chemistry*; D006801:Humans; D009077:Mucins / Q000737:chemistry / Q000378:metabolism*; D058105:Polymerization; D011108:Polymers / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D015202:Protein Engineering
chemical_list: D009077:Mucins; D011108:Polymers; D002918:Chymotrypsin