US 9,539,338 B2 · Polymer-based protein engineering methods to rationally tune enzyme activity, pH-dependence and stability

Abstract

Using a novel water-soluble, active ester amide-containing functionalized controlled radical polymerization initiator, stimuli responsive polymers have been grown from the surface of a protein, exemplified by chymotrypsin or any protein having surface amino acids that will covalently bind to the active ester amide-containing functionalized initiator. It is shown that changes in temperature or pH can change the conformation of the polymer surrounding the enzyme, which in turn enabled the rational tailoring of enzyme activity and stability. This method has afforded an increase in the activity and stability of the enzyme by an order of magnitude at pH's where the enzyme is usually inactive or unstable. Multimodal temperature responsive protein-block copolymer conjugates are described.

Claims39

Dependent claims

A method comprising:

in an aqueous solution, immobilizing an active ester-functionalized amide-containing controlled radical polymerization CRP initiator comprised of the structure
wherein X is a halogen or a chain transfer agent;
R1 is H or alkyl;
R2 is an active ester moiety;
and
n is an integer from 1 to 6,
on each of a plurality of amino binding sites on a protein surface to form a protein-initiator conjugate;
isolating the protein-initiator conjugate;
mixing a first group of monomers having one or more desired properties with the protein-initiator conjugate;
polymerizing the monomers from the protein-initiator conjugate to grow a polymer under controlled radical polymerization conditions to form a protein-polymer conjugate;
and,
isolating the protein-polymer conjugate.

9 more elements

Dependencies are inferred from the claim text.

Facts

Application

14/785,868Filed 22 Apr 2014 · us provisional application

Priority

22 Apr 2013

Published

US 2016/0101190 A114 Apr 2016

Granted

10 Jan 2017

Term ends

Not estimatedThe source records don’t support an estimate.

Classification10 codes

A61K 47/58
The modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule: obtained by reactions only…
A61K 38/4826
Serine endopeptidases: trypsin Chymotrypsin
A61K 47/69
The non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates: the conjugate being characterised by…
C08F 289/00
Macromolecular compounds obtained by polymerising monomers on to macromolecular compounds not provided

Polymer-based protein engineering methods to rationally tune enzyme activity, pH-dependence and stability

Life of this patent

Polymer-based protein engineering methods to rationally tune enzyme activity, pH-dependence and stability

Life of this patent

Abstract

Claims39