Functionalized nanoparticles for enhanced affinity precipitation of proteins

Patent metadata

Field	Value
Publication number	US-11149060-B2
Application number	US-201716076418-A
Country	US
Kind code	B2
Filing date	Feb 17, 2017
Priority date	Feb 19, 2016
Publication date	Oct 19, 2021
Grant date	Oct 19, 2021

Abstract

The present invention provides a nanoparticle capable of binding specifically to a target protein in a solution and precipitating with the target protein out of the solution upon addition of the target protein to the solution. The precipitation may be reversed release the target protein from the nanoparticle, which may be reused for precipitating the target protein. Also provided are a method for purifying a target protein by affinity precipitation using the nanoparticle without chromatography and a method for preparing the nanoparticle.

First claim

What is claimed:

1. A method for purifying a target protein produced by host cells without chromatography, comprising
(a) separating the target protein from the host cells;
(b) exposing the separated target protein of step (a) to a nanoparticle in a first solution to form a complex of the target protein and the nanoparticle, wherein the nanoparticle comprises a fusion protein and a scaffolding domain, wherein the fusion protein is covalently bound to the scaffolding domain, wherein the fusion protein comprises an affinity domain capable of binding specifically the target protein and a stimuli responsive precipitation domain, wherein the nanoparticle is bound specifically to the target protein in the first solution, wherein the scaffolding domain comprises self-assembled proteins and has a diameter of at least 10 nm, wherein the stimuli responsive precipitation domain is an elastin-like polypeptide (ELP) and the scaffolding domain is an E2 core of the pyruvate dehydrogenase complex (E2), wherein the first solution has a salt concentration of 50-200 mM and a pH of 6-9, whereby the complex precipitates out of the first solution;
(c) adding the precipitated complex of step (b) to a second solution having a pH less than 4, whereby the precipitated complex of step (b) is solubilized and the target protein is released from the nanoparticles in the second solution; and
(d) applying a stimulus to the second solution of step (c), wherein the stimulus comprises increasing the salt concentration of the second solution, whereby the solubilized nanoparticle of step (c) precipitates out of the second solution and the released target protein of step (c) is purified from the host cells.

2. The method of claim 1 , wherein the first solution has a temperature of 15-25° C., and wherein the molar ratio of the affinity domain to the target protein in the first solution is in the range of 3:1-6:1.

3. The method of claim 1 , further comprising changing the first solution.

4. The method of claim 1 , further comprising changing temperature of the first solution, salt concentration of the first solution, pH of the first solution or a combination thereof.

5. The method of claim 1 , wherein the first solution has a temperature of 15-25° C., and wherein the method excludes changing the temperature of the first solution.

6. The method of claim 1 , wherein the first solution has a temperature of 15-25° C., further comprising changing the temperature of the first solution of no more than 10° C.

7. The method of claim 1 , wherein the method excludes changing the salt concentration of the first solution.

8. The method of claim 1 , wherein the salt has a cation selected from the group consisting of ammonium, potassium and sodium.

9. The method of claim 1 , wherein the salt has an anion selected from the group consisting of phosphate, sulfate, carboxylate and chloride.

10. The method of claim 1 , wherein the method excludes changing the pH of the first solution.

11. The method of claim 1 , further comprising changing the pH of the first solution by no more than 5 pH units.

12. The method of claim 1 , wherein the stimulus further comprises a change of pH of the second solution .

13. The method of claim 1 , wherein the stimulus further comprises changing the temperature of the second solution to less than 25° C.

14. The method of claim 1 , wherein the target protein is an antibody selected from the group consisting of immunoglobulin (Ig) types IgG, IgD, IgE, IgA and IgM.

15. The method of claim 1 , wherein the host cells are selected from the group consisting of Chinese hamster ovary (CHO) cells, Escherichia coli cells, Saccharomyces cerevisiae cells, Pichia pastoris cells, and Human embryonic kidney (HEK) cells.

16. The method of claim 1 , wherein the affinity domain is selected from the group consisting of Z-domain, protein A, protein G, protein L, protein M, single-chain variable fragment (scFv) domain, and a combination thereof.

17. The method of claim 1 , wherein the affinity domain is Z-domain.

Assignees

• Chen Wilfred
• Swartz Andrew
• Univ Delaware

Inventors

• Chen Wilfred
• Swartz Andrew

Classifications

• C07K2319/00

  Fusion polypeptide · CPC title

• C07K2319/70

  containing domain for protein-protein interaction · CPC title

• C12Y304/2207

  Sortase A (3.4.22.70) · CPC title

• B82Y5/00

  Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery · CPC title

• C07K14/78

  Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG] · CPC title