Protein chips, preparation and use thereof
View Patent ↗The present invention relates to the field of medicine, in particular of research and diagnosis. It relates more particularly to a novel tool for detecting antibodies in a biological sample originating from a mammal. This tool, which is in the form of a protein chip, can be used in screening for new targets of interest involved in the occurrence of an autoimmune disease, in particular of a disease affecting the nervous system of a mammal, and also in the diagnosis or the monitoring of the progression of such an autoimmune disease. The invention also relates to a method for producing such a tool and also to kits comprising it and enabling its use.
1. A method for preparing a protein chip comprising the following steps:
cloning one or more cDNA(s) of interest encoding one or more membrane proteins expressed in the cells of the nervous system of an animal, and which is (are) involved or suspected of being involved in the occurrence of an encephalopathy associated with neuronal excitability disorders, using an expression vector which allows i) the insertion of a tag, in the same reading frame as the sequence encoding the one or more membrane proteins, and ii) the expression of the one or more membrane proteins fused to said tag,
expressing the one or more membrane proteins encoded by said cDNA(s), fused to said tag, in cells in culture,
solubilizing the expressed one or more membrane proteins using a non-denaturing detergent which allows the solubilization of said one or more membrane proteins while at the same time preserving their conformation and their ability to bind to a support, and
depositing said solubilized one or more membrane proteins on a support, thereby obtaining the chip of interest.
2. The method according to claim 1 , wherein said one or more solubilized membrane proteins are selected from the group consisting of an ion channel protein, a transporter protein, and a membrane receptor protein which regulates the activity of an ion protein channel or of a transporter protein.
3. The method according to claim 1 , wherein said at least one or more membrane proteins comprise a protein selected from SEQ ID NO: 1 to SEQ ID NO: 311.
4. The method according to claim 1 , wherein the one or more membrane proteins comprise a protein selected from the group consisting of a GABA receptor; a glutamate receptor; aquaporin 2 (SEQ ID NO: 307); aquaporin 4 (SEQ ID NO: 309); a potassium channel; a calcium channel; an acetylcholine receptor, a protein associated with voltage gated potassium channels; a glycine receptor; connexin 32 (SEQ ID NO: 310); and connexin 43 (SEQ ID NO: 311).
5. The method of claim 4 , wherein the one or more membrane proteins comprise an N-methyl-D-aspartate (NMDA)-activated glutamate receptor.
6. The method of claim 5 , wherein the NMDA-activated glutamate receptor is the NMDAR1 receptor (SEQ ID NO: 62).
7. The method according to claim 1 , wherein the support upon which the membrane proteins are deposited comprises:
a) at least 100 different membrane proteins of interest;
b) between approximately 200 and approximately 400 different proteins of interest;
c) approximately 250 different proteins of interest; or
d) approximately 300 different proteins of interest,
wherein each protein of interest is optionally associated with one or more auxiliary subunits.
8. The method according to claim 1 , wherein said detergent is selected from the group consisting of Triton X-100, Triton X-114, Nonidet P-40, CHAPS, sodium cholate, sodium deoxycholate, digitonin, sarkosyl NL30 and octylglucoside.
9. The method according to claim 1 , wherein said method does not require an ultracentrifugation step.
10. The method according to claim 1 , wherein said method does not comprise a fractionation step using salts.