Marine bacterial gene LfliZ and use

Patent metadata

Field	Value
Publication number	US-10351857-B2
Application number	US-201615765024-A
Country	US
Kind code	B2
Filing date	Oct 24, 2016
Priority date	Jun 15, 2016
Publication date	Jul 16, 2019
Grant date	Jul 16, 2019

Abstract

It relates to a LfliZ gene and its application. The sequence of gene LfliZ is shown in SEQ ID NO.1. This invention also relates to the recombinant protein LfliZ encoded by LfliZ gene and its application in preparation of 2′, 3′-cNMPs. The recombinant LfliZ protein encoded by gene LfliZ can bind four kinds of 2′, 3′-cNMPs during its expression in Escherichia coli. Four kinds of 2′, 3′-cNMPs can be prepared simultaneously from the recombinant E. coli by extracting the recombinant protein LfliZ. The yield of 2′, 3′-cNMPs reaches 2.5 mg/L fermentation broth by the method in the present invention, indicating that this method has a good application potential.

First claim

What is claimed is:

1. A recombinant vector, containing a gene L fliZ having nucleotide sequence as shown in SEQ ID NO:1.

2. The recombinant vector according to claim 1 , wherein the recombinant vector is constructed with a plasmid pET-22b (+).

3. The recombinant vector according to claim 1 , wherein the recombination vector is transformed into a host cell yields a recombinant cell.

4. The recombinant vector according to claim 3 , wherein the host cell is E. coli.

5. The recombinant vector according to claim 4 , wherein the E. coli is E. coli BL21 (DE3).

6. A process for preparing 2′,3′-cyclic nucleoside monophosphates (2′,3′-cNMPs) by using a gene L fliZ , wherein the gene L fliZ having nucleotide sequence as shown in SEQ ID NO:1; cloning the gene L fliZ into a plasmid as a recombinant vector that is transformed into a host cell that yields as a recombinant cell; culturing the recombinant cell in a fermentation medium; then, preparing the 2′, 3′-cNMPs from the recombinant cell.

7. The process according to claim 6 , wherein the plasmid is pET-22b (+); the host cell is E. coli.

8. The process according to claim 6 , wherein the fermentation medium is LB medium.

9. A process for preparing 2′, 3′-cNMPs by using a gene L fliZ , wherein the gene L fliZ having nucleotide sequence as shown in SEQ ID NO:1; cloning the gene L fliZ into a plasmid as a recombinant vector that is transformed into a host cell that yields as a recombinant cell; culturing the recombinant cell in a fermentation medium; breaking the recombinant cells and purifying a recombinant protein L fliZ having the amino acid sequence shown as SEQ ID NO:2; incubating the recombinant protein L fliZ in a recombinant protein L fliZ solution; and preparing the 2′, 3′-cNMPs from the recombinant protein L fliZ solution.

10. The process according to claim 9 , wherein the plasmid is pET-22b (+); the host cell is E. coli.

11. The process according to claim 9 , wherein the fermentation medium is LB medium.

12. The process according to claim 9 , wherein the recombinant protein L fliZ solution is consisting of 10 mM Tris-HCl, 100 mM NaCl, pH 7.5.

Assignees

• Univ Shandong

Inventors

• Chen Xiulan
• Zhang Yuzhong
• Liu Ang
• Song Xiaoyan
• Yu Yang
• Li Pingyi
• Zhang Xiying
• Shi Mei
• Xie Binbin
• Su Hainan
• Qin Qilong

Classifications

• C12N15/70 Primary

  Vectors or expression systems specially adapted for E. coli · CPC title

• C12P19/32

  having a condensed ring system containing a six-membered ring having two N-atoms in the same ring, e.g. purine nucleotides, nicotineamide-adenine dinucleotide · CPC title

• C12P19/305

  Pyrimidine nucleotides · CPC title

• C07K14/195 Primary

  from bacteria · CPC title

• C07K1/34

  by filtration, ultrafiltration or reverse osmosis · CPC title