Site-specifically labeled transmembrane protein nanodisc, and preparation method and use thereof

Patent metadata

Field	Value
Publication number	US-2024402188-A1
Application number	US-202418676905-A
Country	US
Kind code	A1
Filing date	May 29, 2024
Priority date	May 31, 2023
Publication date	Dec 5, 2024
Grant date	—

Abstract

The application belongs to the technical field of biotechnology, and specifically relates to a site-specifically labeled transmembrane protein nanodisc, and a preparation method and use thereof. The preparation method for a site-specifically labeled transmembrane domain (TMD) as provided in this disclosure involves introducing a cysteine mutation into the TMD gene of a transmembrane protein. This mutated TMD is then conjugated with a chemical compound to achieve site-specifical labeling. Subsequently, the site-specifically labeled TMD and an extracellular domain (ECD) are expressed separately. The TMD is incorporated into a nanodisc, which closely mimics the physiological environment, and then ligated to the ECD. This process results in the formation of a site-specifically labeled transmembrane protein nanodisc.

First claim

1 . A preparation method of a site-specifically labeled transmembrane domain (TMD), comprising the following steps:
introducing a cysteine mutation into a TMD gene fragment encoding a transmembrane protein to obtain a cysteine mutation-containing TMD gene fragment; expressing the cysteine mutation-containing TMD gene fragment to obtain a cysteine mutation site-containing TMD peptide segment; and conjugating a chemical compound to a cysteine mutation site on the cysteine mutation site-containing TMD peptide segment to obtain the site-specifically labeled TMD.

2 . The preparation method according to claim 1 , wherein the transmembrane protein comprises a transmembrane protein containing an extracellular domain (ECD) and a TMD.

3 . A site-specifically labeled TMD nanodisc, comprising a site-specifically labeled TMD and a nanodisc; wherein the nanodisc is wrapped on or inserted into the site-specifically labeled TMD; and the site-specifically labeled TMD is prepared by the preparation method according to claim 1 .

4 . The site-specifically labeled TMD nanodisc according to claim 3 , wherein the nanodisc comprises a phospholipid nanodisc.

5 . A site-specifically labeled transmembrane protein nanodisc, comprising the site-specifically labeled TMD nanodisc according to claim 3 and an ECD.

6 . A preparation method of the site-specifically labeled transmembrane protein nanodisc according to claim 5 , comprising: ligating an amino terminus of the site-specifically labeled TMD nanodisc to a carboxyl terminus of the ECD to obtain the site-specifically labeled transmembrane protein nanodisc.

7 . The preparation method according to claim 6 , wherein the ligating is conducted by enzyme ligation or peptide chain ligation.

8 . The preparation method according to claim 7 , wherein an enzyme for the enzyme ligation is selected from the group consisting of a Sortase and a Butelase.

10 . A method for antibody screening and/or verification, comprising using the site-specifically labeled TMD nanodisc according to claim 3 .

11 . A site-specifically labeled TMD nanodisc, comprising a site-specifically labeled TMD and a nanodisc; wherein the nanodisc is wrapped on or inserted into the site-specifically labeled TMD; and the site-specifically labeled TMD is prepared by the preparation method according to claim 2 .

12 . The site-specifically labeled TMD nanodisc according to claim 11 , wherein the nanodisc comprises a phospholipid nanodisc.

13 . A site-specifically labeled transmembrane protein nanodisc, comprising the site-specifically labeled TMD nanodisc according to claim 11 and an ECD.

14 . A site-specifically labeled transmembrane protein nanodisc, comprising the site-specifically labeled TMD nanodisc according to claim 4 and an ECD.

15 . A site-specifically labeled transmembrane protein nanodisc, comprising the site-specifically labeled TMD nanodisc according to claim 12 and an ECD.

16 . A method for antibody screening and/or verification, comprising using the site-specifically labeled TMD nanodisc according to claim 4 .

17 . A method for antibody screening and/or verification, comprising using the site-specifically labeled transmembrane protein nanodisc according to claim 5 .

18 . A method for antibody screening and/or verification, comprising using a site-specifically labeled transmembrane protein nanodisc prepared by the preparation method according to claim 7 .

19 . A method for antibody screening and/or verification, comprising using a site-specifically labeled transmembrane protein nanodisc prepared by the preparation method according to claim 8 .

Assignees

• Univ Nankai

Inventors

• Cao chan
• Gong Junyuan
• Zhou Xiaohui
• Lin Yifan
• Geng Jialu

Classifications

• G01N33/6854 Primary

  Immunoglobulins · CPC title

• C07K14/705 Primary

  Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)} · CPC title

• C12R2001/19

  Escherichia coli · CPC title

• C12N2800/22

  Vectors comprising a coding region that has been codon optimised for expression in a respective host · CPC title

• C12N9/1205

  Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases · CPC title