title: Tailoring enzyme activity and stability using polymer-based protein engineering.
abstract: Polymer-based protein engineering (PBPE) offers an attractive method to predictably modify and enhance enzyme structure and function. Using polymers that respond to stimuli such as temperature and pH, enzyme activity and stability can be predictably modified without a dependence on molecular biology. Herein, we demonstrate that temperature responsive enzyme-polymer conjugates show increased stability while retaining bioactivity and substrate affinity. The bioconjugates were synthesized using a "grafting from" approach, where polymers were grown from a novel water-soluble initiator on the surface of a protein using atom transfer radical polymerization. Prior to polymer synthesis, the polymerization initiating molecule was covalently attached to surface accessible primary amines (lysine, N-terminal) of chymotrypsin, forming a macroinitiator. Poly(N-isopropylacrylamide) and poly[N,N'-dimethyl(methacryloylethyl) ammonium propane sulfonate] were grown separately from the initiator modified chymotrypsin. Both polymers were selected because of their temperature-dependent conformations. We observed that the enzyme-polymer conjugates retained temperature-dependent changes in conformation while still maintaining enzyme function. The conjugates exhibited dramatic increases in enzyme stability over a wide range of temperatures. We can now predictably manipulate enzyme kinetics and stability using polymer-based protein engineering without the need for molecular biology dependent mutagenesis.
authors.affiliation: Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA 15213, United States.
authors.forename: Chad
authors.initials: C
authors.lastname: Cummings
authors.forename: Hironobu
authors.initials: H
authors.lastname: Murata
authors.forename: Richard
authors.initials: R
authors.lastname: Koepsel
authors.forename: Alan J
authors.initials: AJ
authors.lastname: Russell
mesh_terms: D000180:Acrylic Resins / Q000737:chemistry; D000818:Animals; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism*; D004789:Enzyme Activation; D004795:Enzyme Stability; D006868:Hydrolysis; D007700:Kinetics; D008689:Methacrylates / Q000737:chemistry; D058105:Polymerization; D011108:Polymers / Q000737:chemistry*; D015202:Protein Engineering; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D013696:Temperature
chemical_list: C530265:3-dimethyl(methacryloyloxyethyl)ammonium propane sulfonate; D000180:Acrylic Resins; D008689:Methacrylates; D011108:Polymers; D000644:Quaternary Ammonium Compounds; C052970:poly-N-isopropylacrylamide; D002918:Chymotrypsin
keywords: Atom transfer radical polymerization; Chymotrypsin; Enzyme; Enzyme-polymer conjugates; Protein engineering