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.

Semi-permeable coatings fabricated from comb-polymers efficiently protect proteins in vivo

“…The polymerization of an OEGMA monomer with eight to ten oxyethylene units was initiated from these sites. Growing polymers directly from proteins is a powerful approach for generating complex bioconjugates [21][22][23][24][25] and offers the advantage of producing a series of comparable bio-conjugates differing uniquely in the length of the polymer backbone (n). The length of the polymer backbone was varied by allowing the polymerizations to proceed for different times between 30 min and 4 h. The 19 unique bio-conjugates obtained showed monomodal size-exclusion chromatograms (Fig.…”

Preparation of a dual cored hepatoma-specific star glycopolymer nanogel via arm-first ATRP approach

“…By contrast, polySBMA, one example of zwitterionic polymers, exhibits an UCST in aqueous solution between 16°C and 18°C, which indicates that the homopolymers in a dilute aqueous solution elicit a dramatic change in absorbance. 29 It has been reported previously that solubility of a similarly conjugated thermo-responsive copolymer occurs between the LCST and UCST values of the two polymer segments, as a result of intermolecular and intramolecular electrostatic interactions within the zwitterionic …”

“…The upper critical solution temperature (UCST) or lower critical solution temperature (LCST) in a particular solution condition was the midpoint of the temperature absorption curve. 29,30 The average hydrodynamic diameter of the s-(PLAMA-b-PSBMA)-b-PNIPAM in water was measured by dynamic light scattering (DLS) (Brookhaven BI-200SM goniometer) equipped with a solid state laser source emitting at 532 nm and fitted with an external water bath and thermostat. The volume phase transition temperatures of the nanogels were examined by microcalorimetric analyses.…”

Protein–polymer therapeutics: a macromolecular perspective

“…75 Other thermal responsive polymers, e.g. poly(sulfobetaine methacrylamide) ( pSBAm) 76 and poly-[N,N′-dimethyl(methacryloylethyl) ammonium propane sulfonate] ( pDMAPS), 77 have also been used for enzyme modification. By using two consecutive ATRP reactions from the surface of chymotrypsin, Russell et al could tailor the block copolymer pSBAm-block-pNIPAm to impart a double layered shell responsive to two different temperatures and control enzyme activity in a narrow window.…”

“…By using two consecutive ATRP reactions from the surface of chymotrypsin, Russell et al could tailor the block copolymer pSBAm-block-pNIPAm to impart a double layered shell responsive to two different temperatures and control enzyme activity in a narrow window. 76 These examples demonstrated great potential of using smart polymers to create chemically engineered enzymes with adaptable properties. However, the bulk of these studies are investigated at the in vitro level and the elucidation of the in vivo biological behavior for these smart protein-polymer hybrids is currently in progress.…”