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

Patent metadata

Field	Value
Publication number	US-9539338-B2
Application number	US-201414785868-A
Country	US
Kind code	B2
Filing date	Apr 22, 2014
Priority date	Apr 22, 2013
Publication date	Jan 10, 2017
Grant date	Jan 10, 2017

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.

First claim

The invention claimed is:

1. 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;
R 1 is H or alkyl;
R 2 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.

2. The method recited in claim 1 wherein immobilizing the initiator comprises mixing protein and the initiator in a buffer at a pH of about 8 to 9 and stirring for a period of time sufficient to allow the formation of covalent bonds between the initiator and at least a majority of the amino binding sites.

3. The method recited in claim 1 wherein isolating the protein-initiator conjugate comprises removing unreacted and unattached compounds from the solution.

4. The method recited in claim 3 wherein removing unreacted and unattached compounds from the solution comprises passing the solution through a dialysis membrane.

5. The method recited in claim 4 further comprises lyophilizing the protein-polymer conjugate.

6. The method recited in claim 1 wherein the controlled radical polymerization conditions comprise conditions for one of an atom radical polymerization (ATRP) procedure or a reversible-addition fragmentation chain transfer (RAFT) procedure.

7. The method recited in claim 1 wherein the polymerization is an ATRP procedure and the method further comprises:
mixing the protein-initiator conjugate and monomers in a buffer and removing oxygen from the mixture;
separately adding a deoxygenated ligand to an aqueous copper catalyst solution;
transferring the copper-ligand catalyst solution to the protein-initiator conjugate and monomer mixture; and
stirring at 4-25° C. for a sufficient time to allow polymerization.

8. The method recited in claim 7 wherein removing oxygen from the protein-initiator conjugate and monomer mixture comprises bubbling Ar or N 2 through the mixture.

9. The method recited in claim 7 wherein isolating the protein-polymer conjugate comprises passing the mixture through a dialysis membrane and refrigerating for a period of time sufficient to remove copper-ligand catalyst and unreacted monomer.

10. The method recited in claim 7 wherein X in the initiator structure is one of Br, Cl, or F.

11. The method recited in claim 7 wherein the initiator comprises N-2-bromo-2-methylpropanoyl-β-alanine N′-oxysuccinimide ester.

12. The method recited in claim 7 wherein the initiator comprises N-2-chloro-propanoyl-β-alanine N′-oxysuccinimide ester.

13. The method recited in claim 1 wherein the active ester moiety is selected from the group consisting of N-oxysuccinimde ester, nitrophenyl ester, pentahalophenyl ester wherein the halogen is F or Cl, 1-oxybenzotriazole ester, and 2-oxy-4,6-dimethyloxy-1,3,5-triazine ester.

14. The method recited in claim 1 wherein the polymerization comprises a RAFT procedure and the chain transfer agent comprises a thiocarbonylthio agent.

15. The method recited in claim 1 wherein the protein comprises an enzyme and the initiator covalently binds to a majority of the binding sites on the surface of the enzyme.

16. The method recited in claim 1 wherein the protein comprises an enzyme and the initiator covalently binds to at least 85% of the binding sites on the surface of the enzyme.

17. The method recited in claim 1 wherein the enzyme is selected from the group consisting of chymotrypsin, lysozyme, β-Galactosidase, carbonic anhydrase, glucose oxidase, laccase, and acetylcholinesterase.

18. The method recited in claim 1 wherein the polymer comprises a stimuli responsive polymer that responds to at least one stimulus.

19. The method recited in claim 18 wherein the stimulus is one or both of pH and temperature.

20. The method recited in claim 1 wherein the protein-polymer conjugate comprises chymotrypsin modified through high density attachment of thermo-responsive polymers.

21. The method recited in claim 1 wherein the protein-polymer conjugate formed from the controlled radical polymerization comprises a protein-homopolymer conjugate of different polymer chain lengths and the method further comprises:
following the polymerization of the first group of monomers from the protein-initiator conjugate, mixing a second group of monomers having one or more desirable properties with the protein-homopolymer conjugate under controlled radical polymerization conditions to form a block copolymer.

22. The method recited in claim 21 wherein the block copolymer comprises a dual temperature responsive enzyme-pSBAm-block-pNIPAm conjugate having different polymer chain lengths and molecular weights.

23. The method recited in claim 1 wherein the protein comprises an enzyme and the surface charge of the enzyme is modified by growing cationic pQA from multiple sites on the surface of enzyme.

24. The method recited in claim 1 wherein the polymers grown from the plurality of surface amino sites of the protein core form a high density cationic polymer shell around the protein core.

25. The method recited in claim 1 wherein the chain length of the polymers is controlled by adjusting the molar concentration of the first group of monomers added to the protein initiator conjugate to a desired amount.

26. A macroinitiator comprising:
a water soluble active ester-functionalized amide-containing controlled radical polymerization initiator comprised of the structure

wherein X is a halogen or a chain transfer agent;
R 1 is H or alkyl;
R 2 is an active ester moiety; and
n is an integer from 1 to 6, covalently bound to each of a plurality of surface amino acid residues on a protein.

27. The macroinitiator recited in claim 26 wherein the initiator further covalently binds to the N-terminus of the protein.

28. The macroinitiator recited in claim 26 wherein the initiator is N-2-bromo-2-methylpropanoyl-β-alanine N′-oxysuccinimide ester.

29. The macroinitiator recited in claim 26 wherein the initiator is N-2-chloro-propanoyl-β-alanine N′-oxysuccinimide ester.

30. The macroinitiator recited in claim 26 wherein the active ester moiety is selected from the group consisting of N-oxysuccinimde ester, nitrophenyl ester, pentahalophenyl ester, 1-oxybenzotriazole ester, and 2-oxy-4,6-dimethyloxy-1,3,5-triazine ester.

31. The macroinitiator recited in claim 26 wherein the initiator binds to at least 85% of the lysine residues on the protein.

32. A composition comprising:
a water soluble active ester-functionalized amide-containing controlled radical polymerizati

Assignees

• Russell Alan J
• Koepsel Richard R
• Cummings Chad
• Murata Hironobu
• Univ Carnegie Mellon
• Univ Pittsburgh

Inventors

• Russell Alan J
• Koepsel Richard R
• Cummings Chad
• Murata Hironobu

Classifications

• C08F289/00

  Macromolecular compounds obtained by polymerising monomers on to macromolecular compounds not provided for in groups C08F251/00 - C08F287/00 · CPC title

• C12Y304/21001

  Chymotrypsin (3.4.21.1) · CPC title

• A61K38/4826 Primary

  Trypsin (3.4.21.4) Chymotrypsin (3.4.21.1) · CPC title

• A61K47/58 Primary

  obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin · CPC title

• A61K47/69

  the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit · CPC title