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Rational tailoring of substrate and inhibitor affinity via ATRP polymer-based protein engineering.

Biomacromolecules (2014-06-24)
Hironobu Murata, Chad S Cummings, Richard R Koepsel, Alan J Russell
ABSTRACT

Atom transfer radical polymerization (ATRP)-based protein engineering of chymotrypsin with a cationic polymer was used to tune the substrate specificity and inhibitor binding. Poly(quaternary ammonium) was grown from the surface of the enzyme using ATRP after covalent attachment of a protein reactive, water-soluble ATRP-initiator. This "grafting from" conjugation approach generated a high density of cationic ammonium ions around the biocatalytic core. Modification increased the surface area of the protein over 40-fold, and the density of modification on the protein surface was approximately one chain per 4 nm(2). After modification, bioactivity was increased at low pH relative to the activity of the native enzyme. In addition, the affinity of the enzyme for a peptide substrate was increased over a wide pH range. The massively cationic chymotrypsin, which included up to 2000 additional positive charges per molecule of enzyme, was also more stable at extremes of temperature and pH. Most interestingly, we were able to rationally control the binding of two oppositely charged polypeptide protease inhibitors, aprotinin and the Bowman-Birk trypsin-chymotrypsin inhibitor from Glycine max, to the cationic derivative of chymotrypsin. This study expands upon our efforts to use polymer-based protein engineering to predictably engineer enzyme properties without the need for molecular biology.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Dichloromethane, anhydrous, ≥99.8%, contains 40-150 ppm amylene as stabilizer
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2-Propanol, BioReagent, for molecular biology, ≥99.5%
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2-Propanol, suitable for HPLC, 99.9%
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Dichloromethane, suitable for HPLC, ≥99.8%, contains amylene as stabilizer
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2-Propanol, ACS reagent, ≥99.5%
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Ethyl acetate, ACS reagent, ≥99.5%
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Ethyl acetate, anhydrous, 99.8%
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Glycine, suitable for electrophoresis, ≥99%
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Dichloromethane, contains 40-150 ppm amylene as stabilizer, ACS reagent, ≥99.5%
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Hexane, suitable for HPLC, ≥97.0% (GC)
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Glycine, ReagentPlus®, ≥99% (HPLC)
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Ethyl acetate, suitable for HPLC, ≥99.7%
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Dichloromethane, puriss. p.a., ACS reagent, reag. ISO, ≥99.9% (GC)
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2-Propanol, anhydrous, 99.5%
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Hexane, anhydrous, 95%
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Diethyl ether, contains 1 ppm BHT as inhibitor, anhydrous, ≥99.7%
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2-Propanol, puriss. p.a., ACS reagent, reag. ISO, reag. Ph. Eur., ≥99.8% (GC)
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Hexane, puriss. p.a., ACS reagent, reag. Ph. Eur., ≥99% (GC)
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Isopropyl alcohol, meets USP testing specifications
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