title: Dramatically increased pH and temperature stability of chymotrypsin using dual block polymer-based protein engineering.
abstract: 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.
authors: Department of Biomedical Engineering, Doherty Hall 2100, and ‡Disruptive Health Technology Institute, ICES, 1201 Hamburg Hall, Carnegie Mellon University , 5000 Forbes Avenue, Pittsburgh, Pennsylvania 15213, United States.
authors: Chad
authors: C
authors: Cummings
authors: Hironobu
authors: H
authors: Murata
authors: Richard
authors: R
authors: Koepsel
authors: Alan J
authors: AJ
authors: Russell
mesh_terms: D002918:Chymotrypsin / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008689:Methacrylates / Q000737:chemistry; D058105:Polymerization; D011108:Polymers / Q000737:chemistry*; D015202:Protein Engineering; D012996:Solutions; D013696:Temperature; D014867:Water
chemical_list: D008689:Methacrylates; D011108:Polymers; D012996:Solutions; D014867:Water; D002918:Chymotrypsin