Protein imaging

• US 5,756,717 A
• Filed: 05/24/1995
• Issued: 05/26/1998
• Est. Priority Date: 05/24/1995
• Status: Expired due to Fees

• Current Assignee: Perseptive Biosystems Incorporated (Ecovyst, Inc.), Purdue Research Foundation (The Trustees of Purdue University (d/b/a Purdue University))
• Original Assignee: Perseptive Biosystems Incorporated (Ecovyst, Inc.), Purdue Research Foundation (The Trustees of Purdue University (d/b/a Purdue University))
• Inventors: Nadler, Timothy K., Paliwal, Sandeep K., Varady, Laszlo, Regnier, Fred E.
• Primary Examiner(s): Kight, John
• Assistant Examiner(s): Lee, Howard C.
• Application Number:
  US08/448,822
• Time in Patent Office: 1,098 Days
• Field of Search: 536/123, 536/123.1, 536/123.13, 536/124, 536/103, 536/112, 530/344, 530/345, 530/361, 530/368, 530/369, 530/412, 530/413, 530/416, 530/417
• US Class Current: 536/123.1
• CPC Class Codes: B01D 15/3852 using imprinted phases or molecular recognition; using imprinted phases
• CPC Class Codes: B01J 20/26 Synthetic macromolecular compounds
• CPC Class Codes: B01J 20/268 Polymers created by use of a template, e.g. molecularly imprinted polymers
• CPC Class Codes: B01J 20/285 based on polymers
• CPC Class Codes: B01J 20/291 Gel sorbents
• CPC Class Codes: C07K 1/22 Affinity chromatography or related techniques based upon selective absorption processes

Abstract

Disclosed are specifically designed binding, "protein imaged" sorbents which reversibly bind with high specificity and affinity a preselected macromolecule, specifically a protein. The sorbents define one or more cavities which have a binding surface complementary in shape to the molecular surface of the macromolecule and a plurality of positively and negatively charged chemical moieties spatially distributed in a mirror image and charge inverse of a subset of the ionizable groups on the molecular surface of the macromolecule. Also disclosed are methods of producing such sorbents, useful over a range of conditions, for both preparative and analytical chromatographic separations or for use in various types of analyses.

Claims

1. A composition of matter which selectively binds a preselected polypeptide chain having a plurality of ionizable groups spaced about a molecular surface thereof, the composition comprising:

a shape-retaining porous gel defining a cavity, the cavity having a binding surface complementary in shape to the molecular surface of the polypeptide chain and having a plurality of positively and negatively charged chemical moieties spatially distributed in a mirror image and charge inverse of a subset of the ionizable groups on the molecular surface of the polypeptide chain, the shape-retaining porous gel having a binding affinity for the polypeptide chain of at least 10⁵ M^-1.

3. The composition of claim 1 or 2, wherein the positively charged chemical moiety is selected from the group of chemical moieties consisting of a primary amine, secondary amine, tertiary amine, and quaternary amine.

4. The composition of claim 3, wherein the positively charged chemical moiety is a diethyaminoethyl moiety.

5. The composition of claim 1 or 2, wherein the negatively charged chemical moiety is selected from the group of chemical moieties consisting of a carboxylate, sulfonate, phosphate, and phosphonate.

6. The composition of claim 5, wherein the negatively charged chemical moiety is a carboxylmethyl moiety.

7. The composition of claim 1 or 2, wherein the shape-retaining porous gel is a naturally occurring polymer.

8. The composition of claim 7, wherein the shape-retaining porous gel is agarose.

9. The composition of claim 8, wherein the shape-retaining porous gel comprises a mixture of neutral agarose, diethylamino ethyl agarose, and carboxymethyl agarose.

10. The composition of claim 1 or 2, wherein the shape-retaining porous gel defines pores having an internal diameter in a range of from about 200 Å

to about 1000 Å

thereby to permit the polypeptide chain to be removed from the gel.

11. The composition of claim 10, wherein the pores have an internal diameter in the range from about 300 Å

to about 600 Å

.

12. A chromatography matrix comprising the composition of claim 1 or 2.

25. In a method of separating solutes in a mixture comprising passing the mixture through a chromatography matrix which differentially binds individual solutes in the mixture and then desorbing solutes bound to the chromatography matrix, the improvement comprising:

a) providing the chromatography matrix of claim 12 wherein the preselected polypeptide chain is a target solute in the mixture; and

b) passing the mixture through the chromatography matrix thereby to bind preferentially the target solute to the shape-retaining porous gel.

26. In a method for selectively binding a solute in a mixture, the improvement comprising:

a) providing the composition of claim 1 or 2, wherein the preselected polypeptide chain is a target solute in the mixture; and

b) admixing the composition with the mixture under conditions such that the target solute binds selectively with the composition.

2. A composition of matter which selectively binds a preselected polypeptide chain having a plurality of ionizable groups spaced about a molecular surface thereof, the composition comprising:

a) a rigid particle defining pores; and

b) disposed within the pores, a shape-retaining porous gel defining a cavity, the cavity having a binding surface complementary in shape to the molecular surface of the polypeptide chain and having a plurality of positively and negatively charged chemical moieties spatially distributed in a mirror image and charge inverse of a subset of the ionizable groups on the molecular surface of the polypeptide chain, the shape-retaining porous gel having a binding affinity for the polypeptide chain of at least 10⁵ M^-1.

13. A method for producing a shape-retaining porous gel which selectively binds a preselected polypeptide chain having a plurality of ionizable groups spaced about a molecular surface thereof, the method comprising the steps of:

a) providing an aqueous solution of a conformationally compliant polymer matrix material at a pH and at a temperature that permits formation of a shape-retaining porous gel, the matrix material having disposed therein positively charged and negatively charged chemical moieties;

b) admixing the matrix material with the preselected polypeptide chain to produce a matrix material-polypeptide chain mixture under conditions to produce electrostatic pairing between at least a subset of the chemical moieties in the matrix material and at least a subset of the ionizable groups on the molecular surface of the polypeptide chain;

c) inducing the matrix material to form a shape-retaining porous gel; and

d) removing the polypeptide chain disposed within the shape-retaining porous gel thereby to produce within the gel an image of the disassociated polypeptide chain, the image having a stereochemical shape complementary to the molecular surface of the disassociated polypeptide chain and having spatially distributed chemical moieties in a mirror image and charge inverse of the subset of the ionizable groups on the molecular surface of the disassociated polypeptide chain.

14. The method of claim 13 comprising the additional step of:

prior to step c, combining the mixture of step b with rigid particles defining a pore under conditions to permit the mixture to enter the pore.

15. The method of claim 13 or 14, wherein the positively charged chemical moiety is selected from the group of chemical moieties consisting of a primary amine, secondary amine, tertiary amine, and quaternary amine.

16. The method of claim 15, wherein the positively charged chemical moiety is a diethyaminoethyl moiety.

17. The method of claim 13 or 14, wherein the negatively charged chemical moiety is selected from the group of chemical moieties consisting of a carboxylate, sulfonate, phosphate, and phosphonate.

18. The method of claim 17, wherein the negatively charged chemical moiety is a carboxylmethyl moiety.

19. The method of claim 13 or 14, wherein the matrix material is a naturally occurring polymer.

20. The method of claim 19, wherein the matrix material is agarose.

21. The method of claim 20, wherein the matrix material comprises a mixture of neutral agarose, diethylaminoethyl agarose, and carboxymethyl agarose.

22. The method of claim 13 or 14, wherein step d is conducted by adding to the shape-retaining porous gel a salt solution of ionic strength sufficient to disassociate the polypeptide chain from the gel.

23. A shape-retaining porous gel produced by the method of:

a) providing an aqueous solution of a conformationally compliant polymer matrix material at a pH and at a temperature that permits formation of a shape-retaining porous gel, the matrix material having disposed therein positively charged and negatively charged chemical moieties;

b) admixing the matrix material with the preselected polypeptide chain having a plurality of ionizable groups spaced about a molecular surface thereof under conditions to produce electrostatic pairing between at least a subset of the chemical moieties in the matrix material and at least a subset of the ionizable groups on the molecular surface of the polypeptide chain;

c) inducing the matrix material to form a shape-retaining porous gel; and

d) removing the polypeptide chain disposed within the shape-retaining porous gel thereby to produce within the gel an image of the disassociated polypeptide chain, the image having a stereochemical shape complementary to the molecular surface of the disassociated polypeptide chain and having spatially distributed chemical moieties in a mirror image and charge inverse of the subset of the ionizable groups on the molecular surface of the disassociated polypeptide chain.

24. A rigid particle produced by the method of:

a) providing an aqueous solution of a conformationally compliant polymer matrix material at a pH and at a temperature that permits formation of a shape-retaining porous gel, the matrix material having disposed therein positively charged and negatively charged chemical moieties;

b) admixing the matrix material with the preselected polypeptide chain having a plurality of ionizable groups spaced about a molecular surface thereof under conditions to produce electrostatic pairing between at least a subset of the chemical moieties in the matrix material and at least a subset of the ionizable groups on the molecular surface of the polypeptide chain;

c) combining the product of step b) with a rigid particle defining a pore under conditions to permit the mixture to enter the pore of the rigid particle;

d) inducing the matrix material to form a shape-retaining porous gel within the pore of the rigid particle; and

e) removing polypeptide chain disposed within the shape-retaining porous gel thereby to produce within the gel an image of the disassociated polypeptide chain, the image having a stereochemical shape complementary to the molecular surface of the disassociated polypeptide chain and having spatially distributed chemical moieties in a mirror image and charge inverse of the subset of the ionizable groups on the molecular surface of the disassociated polypeptide chain.