IP Library Granted Patent US 10,533,161
Granted Patent B2
US 10,533,161 · App. 15/306,137 · Granted Jan 14, 2020

NADPH oxidase proteins

Inventors: Jérôme Dupuy (Saint Quentin Sur Isère, FR); Christine Hajjar (Bekaa, LB); Mickaël Cherrier (Tullins, FR); Franck Fieschi (Vif, FR)
Assignees: UNIVERSITE GRENOBLE ALPES; CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE
C12N9/0036C12Q1/28C12Y106/03001G01N33/52G01N2333/90209
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Quick Facts
Patent No.
US 10,533,161
App. No.
15/306,137
Granted
Jan 14, 2020
Kind
B2
Abstract

A NADPH oxidase (Nox) protein includes a domain including 3 to 7 transmembrane helices, and an amino acid sequence including at least 2 bis-histidyl motifs, wherein each of the bis-histidyl motifs consists of 2 histidine residues separated by 12, 13 or 14 amino acid residues. The Nox protein may be produced by a method that includes solubilizing a solution of cells expressing the Nox protein using a detergent from the maltoside family to suspend the Nox protein in the solution.

Claims (29)

1. A NADPH oxidase (Nox) protein comprising:

(a) a domain comprising from 3 to 7 transmembrane helices;

(b) an amino acid sequence comprising at least 2 bis-histidyl motifs, wherein each of the bis-histidyl motifs consists of 2 histidine residues separated by 12, 13 or 14 amino acid residues;

(c) at least one first motif consisting of the sequence:

(SEQ ID NO: 1)

[G/S/A]-[Q/D]-F-[A/V/T/L/F]-[F/Y/W/L/R]-[L/V/I/F/

W];

(d) at least one second motif consisting of the sequence:

(SEQ ID NO: 2)

[P/A/S/E/F]-H-[P/S/A]-F-[T/S]-[L/I/M/V];

(e) at least one third motif consisting of the sequence:

[K/R]-X-X-G-[D/G]-X-[T/S]  (SEQ ID NO: 3),

wherein X represents any naturally occurring amino acid; and

(f) at least one fourth motif consisting of the sequence:

(SEQ ID NO: 4)

G-[I/S/V/L]-G-[V/I/A/F]-[T/A/S]-[P/Y/T/A],

wherein the Nox protein is produced in a host cell comprising an expression vector comprising a nucleic acid sequence selected from the group consisting of the sequences SEQ ID NOs: 365 to 369.

2. The Nox protein of claim 1 , wherein said first motif is selected from the group consisting of the sequences SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10.

3. The Nox protein of claim 1 , wherein said second motif is selected from the group consisting of the sequences, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14.

4. The Nox protein of claim 1 , wherein said third motif is selected from the group consisting of the sequences, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.

5. The Nox protein of claim 1 , wherein said fourth motif is selected from the group consisting of the sequences SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23.

6. The Nox protein of claim 1 , wherein the Nox protein comprises NADPH oxidase activity which catalyzes the production of reactive oxygen species (ROS) in the presence of an electron donor.

7. The Nox protein of claim 6 , wherein said NADPH oxidase activity is capable of being detected in a sample comprising said protein via a reaction showing production of superoxide ion.

8. The Nox protein of claim 1 , wherein said host cell is a bacterium.

9. A method for producing the Nox protein of claim 1 , the method comprising the steps of:

(a) providing a solution of cells comprising said Nox protein in the cellular membranes of said cells; and

(b) using at least one detergent from the maltoside family, solubilizing the cell membranes to place said Nox protein in suspension in said solution.

10. The method of claim 9 , wherein said at least one detergent is a dodecyl-maltoside.

11. The method of claim 9 , wherein said at least one detergent is selected from the group consisting of: n-Dodecyl-β-D-Maltopyranoside; n-Decyl-α-D-Maltopyranoside; 2,6-Dimethyl-4-Heptyl-β-D-Maltopyranoside; HEGA-11; n-Hexyl-β-D-Maltopyranoside; n-Hexadecyl-β-D-Maltopyranoside; n-Nonyl-β-D-Maltopyranoside; n-Octyl-β-D-Maltopyranoside; 2-Propyl-1-Pentyl-β-D-Maltopyranoside; n-Tetradecyl-β-D-Maltopyranoside; n-Tridecyl-β-D-Maltopyranoside; n-Undecyl-β-D-Maltopyranoside; n-Undecyl-α-D-Maltopyranoside; ω-Undecylenyl-β-D-Maltopyranoside; n-Decyl-β-D-Thiomaltopyranoside; n-Dodecyl-β-D-Thiomaltopyranoside; n-Octyl-β-D-Thiomaltopyranoside; n-Nonyl-β-D-Thiomaltopyranoside; n-Undecyl-β-D-Thiomaltopyranoside; Lauryl Maltose Neopentyl Glycol (MNG3); Decyl Maltose Neopentyl Glycol; CYMAL-5 Neopentyl Glycol; CYMAL-6 Neopentyl Glycol; CYMAL-7 Neopentyl Glycol; CYMAL-1; CYMAL-2; CYMAL-3; CYMAL-4; CYMAL-5; CYMAL-6; CYMAL-7; Octyl Maltoside, Fluorinated; BisMalt-18; BisMalt-20; BisMalt-22; BisMalt-24; BisMalt-28; n-Dodecyl-d25-β-D-Maltopyranoside; Decyl-β-D-Selenomaltoside; Dodecyl-β-D-Selenomaltoside; Octyl-β-D-Selenomaltoside; and Undecyl-β-D-Selenomaltoside.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Dec 15, 2016
From: DUPUY, JÉRÔME; HAJJAR, CHRISTINE; CHERRIER, MICKAËL; FIESCHI, FRANCK
To: UNIVERSITE GRENOBLE ALPES; CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE
Reel/Frame 040739/0320 →
Priority Claims (1)
FR 1453616 · Apr 22, 2014 · national
Continuity (1)
Related Publication 20170088826A1 · Mar 30, 2017