Method for using nanoparticles as nucleation agents for the crystallization of proteins

Patent metadata

Field	Value
Publication number	US-9932366-B2
Application number	US-201615287444-A
Country	US
Kind code	B2
Filing date	Oct 6, 2016
Priority date	Oct 7, 2015
Publication date	Apr 3, 2018
Grant date	Apr 3, 2018

Abstract

The present invention relates to a method for the crystallization of protein using nanoparticles as nucleation agents. Precisely, the inventors performed the crystallization of such proteins as Bacillus subtilis YesR, chicken egg white lysozyme, bovine serum albumin, Alicyclobacillus acidocaldarius acetyl-CoA carboxylase, and Listeria monocytogenes hypothetical protein which have his-tag at amino terminal by using gold nanoparticles in diverse sizes and shapes in the presence of Ni 2+ ions. As a result, it was confirmed that the chance of successful crystallization was higher with the gold nanoparticles than without the gold nanoparticles and the various crystallization conditions were successfully screened. Therefore, the method for inducing nucleation of the invention can be advantageously used for the disclosure of protein structure by increasing the chance of successful crystallization of protein.

First claim

What is claimed is:

1. A method for inducing nucleation for the crystallization of protein which includes the step of mixing the nanoparticles coated with carboxyl group with the protein having his-tag in the presence of Ni 2+ ions.

2. The method for inducing nucleation for the crystallization of protein according to claim 1 , wherein the nanoparticles are selected from the group consisting of platinum nanoparticles, silver nanoparticles, magnetic nanoparticles, polymer nanoparticles, and gold nanoparticles.

3. The method for inducing nucleation for the crystallization of protein according to claim 2 , wherein the nanoparticles are gold nanoparticles.

4. The method for inducing nucleation for the crystallization of protein according to claim 1 , wherein the surface of nanoparticle is coated with carboxyl group using a carboxyl group-rich organic molecule.

5. The method for inducing nucleation for the crystallization of protein according to claim 4 , wherein the carboxyl group-rich organic molecule is selected from the group consisting of citrate, mercapundecanoic acid (MUA), mercaptopropionic acid (MPA), poly acrylic acid (PAA), glutathione (GSH), thioglycolic acid (TGA), cysteine, mercaptobenzoic acid, D-penicillamine, and dihydrolipoic acid (DHLA).

6. The method for inducing nucleation for the crystallization of protein according to claim 1 , wherein the nanoparticle is in one of those shapes selected from the group consisting of sphere, rod, and star shapes.

7. The method for inducing nucleation for the crystallization of protein according to claim 6 , wherein the nanoparticles in the shape of sphere are 5˜100 nm in diameter, those in the shape of rod are 36˜55.5 nm in length and 15 nm in diameter, and those in the shape of star are 15˜230 nm in diameter.

8. The method for inducing nucleation for the crystallization of protein according to claim 1 , wherein the number of nanoparticles used for the crystallization of protein is 10 3 ˜10 7 .

9. The method for inducing nucleation for the crystallization of protein according to claim 1 , wherein the protein is selected from the group consisting of those proteins having his-tag.

10. The method for inducing nucleation for the crystallization of protein according to claim 1 , wherein the count rate of proteins and nanoparticles are 1:10 7 ˜1:10 13 .

11. A method for inducing selective binding of protein for the crystallization of protein which contains the step of mixing the nanoparticles coated with carboxyl group and the proteins having his-tag in the presence of Ni 2+ ions.

12. A kit for the crystallization of protein containing the followings:
1) a resin mixed with nanoparticles coated with carboxyl group containing Ni 2+ ; and
2) a protein having his-tag on the surface thereof.

Assignees

• Univ Seoul Ind Coop Found

Inventors

• Choe Jungwoo
• Choi Inhee

Classifications

• C07B2200/13

  Crystalline forms, e.g. polymorphs · CPC title

• C07K14/32

  from Bacillus (G) · CPC title

• C30B7/00

  Single-crystal growth from solutions using solvents which are liquid at normal temperature, e.g. aqueous solutions (from molten solvents C30B9/00; by normal or gradient freezing C30B11/00; under a protective fluid C30B27/00) · CPC title

• C07K1/306 Primary

  by crystallization · CPC title

• C30B29/58

  Macromolecular compounds · CPC title