Method for endogenously extracting mycobacterium smegmatis protein nanocage

Patent metadata

Field	Value
Publication number	US-2026002186-A1
Application number	US-202418826473-A
Country	US
Kind code	A1
Filing date	Sep 6, 2024
Priority date	Jul 1, 2024
Publication date	Jan 1, 2026
Grant date	—

Abstract

A method for endogenously extracting a Mycobacterium smegmatis protein nanocage is provided. The method includes the following steps: introducing a recombinant plasmid containing a CFP29 gene and a 1×Flag affinity tag into a Mycobacterium smegmatis strain to obtain a recombinant Mycobacterium smegmatis strain; extracting a total protein solution of the recombinant Mycobacterium smegmatis strain, and subjecting the total protein solution of the recombinant Mycobacterium smegmatis strain to Flag tag affinity column chromatography purification to obtain a crude extract of the Mycobacterium smegmatis protein nanocage; and subjecting the crude extract of the Mycobacterium smegmatis protein nanocage to gel exclusion chromatography purification to obtain a pure product of the Mycobacterium smegmatis protein nanocage. The method is simple to operate and convenient to implement during extraction and purification, and an obtained Mycobacterium smegmatis background protein nanocage has a high yield, excellent purity, and stable properties.

First claim

What is claimed is:

1 . A method for endogenously extracting a Mycobacterium smegmatis protein nanocage, comprising the following steps:
introducing a recombinant plasmid containing a CFP29 gene and a 1×Flag affinity tag into a Mycobacterium smegmatis strain to obtain a recombinant Mycobacterium smegmatis strain; extracting a total protein solution of the recombinant Mycobacterium smegmatis strain, and subjecting the total protein solution of the recombinant Mycobacterium smegmatis strain to Flag tag affinity column chromatography purification to obtain a crude extract of the Mycobacterium smegmatis protein nanocage; and subjecting the crude extract of the Mycobacterium smegmatis protein nanocage to gel exclusion chromatography purification to obtain a pure product of the Mycobacterium smegmatis protein nanocage.

2 . The method according to claim 1 , wherein the recombinant plasmid is pUC19-CFP29-Str-Flag.

3 . The method according to claim 1 , wherein a construction process of the recombinant plasmid comprises: transferring the CFP29 gene, an upstream 806 bp nucleotide sequence of the CFP29 gene, a GGS-Linker, the 1×Flag affinity tag, a streptomycin selection marker, and a downstream 1,578 bp nucleotide sequence of the CFP29 gene into a vector plasmid.

4 . The method according to claim 3 , wherein the upstream 806 bp nucleotide sequence of the CFP29 gene is shown in SEQ ID NO: 5, and the downstream 1,578 bp nucleotide sequence of the CFP29 gene is shown in SEQ ID NO: 6.

5 . The method according to claim 1 , wherein the Flag tag affinity column chromatography purification is conducted on a chromatographic column filled with an Anti-Flag filler; and the gel exclusion chromatography purification is conducted on a chromatographic column of Superose 6 increase 10/300 gel exclusion.

6 . The method according to claim 1 , wherein the Flag tag affinity column chromatography purification comprises impurity elution and elution that are conducted in sequence; an impurity eluent for the impurity elution comprises 3-(N-morpholino) propanesulfonic acid, NaCl, and a detergent; and a eluent for the elution comprises the 3-(N-morpholino) propanesulfonic acid, the NaCl, and a 1×Flag peptide.

7 . The method according to claim 6 , wherein the detergent comprises polyoxyethylene lauryl ether (POELE).

8 . The method according to claim 1 , wherein an equilibrium buffer for the gel exclusion chromatography purification comprises 3-(N-morpholino) propanesulfonic acid and NaCl.

9 . The method according to claim 8 , wherein the 3-(N-morpholino) propanesulfonic acid has a molar concentration of 20 mmol/L to 25 mmol/L and the NaCl has a molar concentration of 100 mmol/L to 110 mmol/L in the equilibrium buffer.

10 . The method according to claim 1 , wherein a process of extracting the total protein solution of the recombinant Mycobacterium smegmatis strain comprises: culturing the recombinant Mycobacterium smegmatis strain, collecting a resulting recombinant Mycobacterium smegmatis bacterial cell, resuspending the recombinant Mycobacterium smegmatis bacterial cell with a resuspension reagent, conducting high-pressure cell disruption, and collecting a supernatant obtained by centrifugation to obtain the total protein solution.

11 . The method according to claim 2 , wherein a construction process of the recombinant plasmid comprises: transferring the CFP29 gene, an upstream 806 bp nucleotide sequence of the CFP29 gene, a GGS-Linker, the 1×Flag affinity tag, a streptomycin selection marker, and a downstream 1,578 bp nucleotide sequence of the CFP29 gene into a vector plasmid.

12 . The method according to claim 11 , wherein the upstream 806 bp nucleotide sequence of the CFP29 gene is shown in SEQ ID NO: 5, and the downstream 1,578 bp nucleotide sequence of the CFP29 gene is shown in SEQ ID NO: 6.

Assignees

• Univ Nankai

Inventors

• Gong hongri
• Zhao Ziqing

Classifications

• C12N2800/101

  for bacteria · CPC title

• C12N15/74 Primary

  Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora · CPC title

• C12P21/02 Primary

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C12R2001/34

  Mycobacterium smegmatis · CPC title

• C07K14/35

  from Mycobacteriaceae (F) · CPC title