HIGH-PRESSURE REFOLDING OF PROTEINS IN THE PRESENCE OF BINDING PARTNERS
Methods for producing biologically active protein from aggregated and/or denatured proteins which comprise subjecting the protein to high hydrostatic pressure in the presence of a ligand or specific binding agent are disclosed. The ligand can be a macromolecule, such as another protein, a nucleic acid, or other macromolecules, or the ligand can be a small organic molecule.
1 . A method for producing biologically active protein from a mixture comprising aggregated or denatured protein, comprising:
a) adding a specific binding agent for the biologically active protein to the mixture;
b) subjecting the mixture to high hydrostatic pressure for a period of time sufficient to form biologically active protein; and
c) reducing the pressure to atmospheric pressure.
2 . The method of claim 1 , wherein the specific binding agent is a small organic molecule.
3 . The method of claim 2 , wherein the small organic molecule is a rigid molecule.
4 . The method of claim 2 , wherein the small organic molecule is a flexible molecule.
5 . The method of claim 1 , wherein the specific binding agent is a polypeptide.
6 . The method of claim 1 , wherein the specific binding agent is a nucleic acid molecule.
7 . A method for producing biologically active protein from a mixture comprising aggregated or denatured protein, comprising:
a) adding a homopolymer or non-naturally-occurring polymer which binds to the biologically active protein to the mixture;
b) subjecting the mixture to high hydrostatic pressure for a period of time sufficient to form biologically active protein; and
c) reducing the pressure to atmospheric pressure.
8 . The method of claim 7 , wherein the homopolymer or non-naturally occurring polymer binds specifically to the biologically active protein.
9 . The method of claim 7 , wherein the homopolymer or non-naturally occurring polymer binds preferentially to the biologically active protein over inactive, denatured, or aggregated protein.
10 . The method of claim 9 , wherein the homopolymer or non-naturally occurring polymer binds preferentially to the biologically active protein via electrostatic interaction.
11 . The method of claim 9 , wherein the homopolymer or non-naturally occurring polymer binds preferentially to the biologically active protein via hydrophobic interaction.
12 . The method of claim 9 , wherein the homopolymer or non-naturally occurring polymer is a homopolymer.
13 . The method of claim 9 , wherein the homopolymer or non-naturally occurring polymer is a non-naturally occurring polymer.
14 . The method of claim 9 , wherein the homopolymer or non-naturally occurring polymer is dextran sulfate.
15 . A method for producing biologically active protein from a mixture comprising a first aggregated or denatured protein, comprising:
a) adding a second aggregated or denatured protein to the mixture; and
b) subjecting the mixture to high hydrostatic pressure for a period of time sufficient to form biologically active protein, wherein said first and second aggregated or denatured proteins specifically interact under high pressure; and
c) reducing the pressure to atmospheric pressure.
16 . The method of claim 15 , further comprising:
d) separating the first and second proteins.
17 . The method of claim 16 , wherein the separating of the first and second proteins is performed by affinity chromatography, HPLC, dialysis, ion exchange chromatography, size exclusion chromatography, reverse-phase chromatography, ammonium sulfate precipitation, or electrophoresis.
18 . The method of claim 15 , wherein one of the first or second proteins is a chaperone protein.
19 . The method of claim 15 , wherein the first and second proteins continue to interact after the pressure is reduced to atmospheric pressure.
20 . The method of claim 19 , wherein the first and second proteins form a heterodimer in their biologically active state.
21 . The method of claim 19 , wherein one of the first and second proteins is an enzyme and the other is a substrate for the enzyme.
22 . The method of claim 19 , wherein one of the first and second proteins is an enzyme and the other is an inhibitor of the enzyme.
23 . The method of claim 19 , wherein one of the first and second proteins is an enzyme and the other is a regulator or modulator of the enzyme.
24 . The method of claim 19 , wherein one of the first and second proteins is a receptor and the other is a ligand for the receptor.
25 . The method of claim 24 , wherein one of the first and second proteins is a receptor and the other is an agonist for the receptor.
26 . The method of claim 24 , wherein one of the first and second proteins is a receptor and the other is an antagonist for the receptor.