METHOD OF PRODUCING MONOMERIC CARD IN BACTERIA

• US 20190338265A1
• Filed: 01/10/2019
• Published: 11/07/2019
• Est. Priority Date: 05/02/2018
• Status: Active Grant

• Current Assignee: Korea Advanced Institute of Science and Technology
• Original Assignee: Korea Advanced Institute of Science and Technology
• Inventors: CHUNG, Hak Suk, AN, Jinsu, YANG, Eun Gyeong, KIM, So Yeon
• Granted Patent: US 10,640,756 B2
• Time in Patent Office: Days
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 9/50 Proteinases , e.g. Endopeptidases (3.4.21-3.4.25)
• CPC Class Codes: C12Y 304/22057 Caspase-4 (3.4.22.57)
• CPC Class Codes: C12Y 304/22064 Caspase-11 (3.4.22.64)

Abstract

Provided is a method of producing monomeric caspase activation and recruitment domains (CARDs) in bacteria. According to this method, CARD domains may be easily produced in a monomeric form. Monomeric CARDs may be applied to studies on the mechanism of action of caspase, the development of new drugs, detection and elimination of endotoxins, development of cosmetics, etc.

Claims

1. A method of producing monomeric caspase activation and recruitment domains (CARDs) in bacteria, the method comprising:

introducing a vector comprising a polynucleotide encoding a caspase (Casp) protein into bacteria to prepare transformed bacteria;

culturing the transformed bacteria to induce expression of caspase;

lysing the cultured bacteria to obtain a lysate; and

obtaining monomeric CARDs from the lysate.

2. The method of claim 1, wherein the caspase is selected from the group consisting of caspase-1, caspase-2, caspase-4, caspase-5, caspase-9, caspase-11, caspase-12, and caspase-13.

3. The method of claim 1, wherein the caspase is a wild-type caspase or a mutant caspase.

4. The method of claim 3, wherein the mutant caspase has a substitution (C258A) of alanine (A) for cysteine (C) which is an amino acid at position 258 from the N-terminus thereof.

5. The method of claim 1, wherein the caspase is a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:

1, 7, 9, and 13.

6. The method of claim 1, wherein the vector comprises a polynucleotide encoding an affinity tag.

7. The method of claim 6, wherein the affinity tag is a polyhistidine tag, GST, MBP, NusA, thioredoxin, ubiquitin, FLAG, BAP, STREP, CBP, CBD, hemagglutinin (HA), S-tag, Small Ubiquitin-like Modifier (SUMO), or a combination thereof.

8. The method of claim 1, wherein the bacteria are Escherichia coli.

9. The method of claim 8, wherein the Escherichia coli is selected from the group consisting of C41(DE3), ClearColi BL21(DE3), and CMR300.

10. The method of claim 1, wherein the bacteria are cultured in a medium comprising an antibiotic.

11. The method of claim 1, wherein the lysing of the cultured bacteria is performed in the presence of a surfactant.

12. The method of claim 11, wherein the surfactant is selected from the group consisting of Tween 20, Tween 80, Triton X-100, Triton X-114, NP-40, Brij-35, Brij-58, octyl glucoside, octyl thioglucoside, n-Dodecyl β

-D-maltoside (DDM), CHAPS, and CHAPSO.

13. The method of claim 1, wherein the lysing of the bacteria is performed by sonication.

14. The method of claim 1, further comprising heating the lysate.

15. The method of claim 1, wherein the lysate is subjected to a method selected from the group consisting of affinity chromatography, size-exclusion chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, and high-performance liquid chromatography (HPLC).

16. The method of claim 15, wherein the affinity chromatography is nickel (Ni)-affinity chromatography.

17. The method of claim 15, wherein the size-exclusion chromatography comprises use of a gel selected from the group consisting of Superdex, Superose, Sephacryl, Sephadex, PL gel, cross-linked polyacrylamide, agarose gel, and Styragel.