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  • Tailoring enzyme activity and stability using polymer-based protein engineering.

Tailoring enzyme activity and stability using polymer-based protein engineering.

Biomaterials (2013-07-16)
Chad Cummings, Hironobu Murata, Richard Koepsel, Alan J Russell
摘要

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.

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Sigma-Aldrich
α-糜蛋白酶 来源于牛胰腺, Type II, lyophilized powder, ≥40 units/mg protein
Sigma-Aldrich
N-异丙基丙烯酰胺, Mn ~40,000
Sigma-Aldrich
α-糜蛋白酶 来源于牛胰腺, (TLCK treated to inactivate residual tryspin activity), Type VII, essentially salt-free, lyophilized powder, ≥40 units/mg protein
Sigma-Aldrich
α-糜蛋白酶 来源于牛胰腺, Type I-S, essentially salt-free, lyophilized powder
Sigma-Aldrich
α-糜蛋白酶 来源于牛胰腺, suitable for protein sequencing, salt-free, lyophilized powder
Sigma-Aldrich
α-糜蛋白酶 来源于牛胰腺, ≥40 units/mg protein, vial of 5 mg
Sigma-Aldrich
羧酸封端的聚(N-异丙基丙烯酰胺), average Mn 10,000
Sigma-Aldrich
α-糜蛋白酶 来源于人类胰腺, lyophilized powder
Sigma-Aldrich
Poly(N-isopropyl acrylamide), NHS ester end functionalized, average Mn 5,000
Sigma-Aldrich
α-Chymotrypsin−Agarose from bovine pancreas, lyophilized powder, 2,000-3,500 units/g agarose (One ml gel will yield 65-120 units)