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Lipid binding protein-antigen capturing module compound and preparing method and application thereof

A technology for binding proteins and complexes, applied in the biological field, which can solve the problems of extraction of membrane proteins, low assembly efficiency, unsatisfactory uniformity, etc.

Active Publication Date: 2019-08-13
ASSEMBLY MEDICINE LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, the preparation of native antigens requires the assembly of MSP or Saposin, lipids, and membrane proteins, and membrane proteins need to be highly purified
The bottleneck of the current native antigen lies in the low assembly efficiency, unsatisfactory uniformity and the inability to directly extract membrane proteins from living cell membranes

Method used

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  • Lipid binding protein-antigen capturing module compound and preparing method and application thereof
  • Lipid binding protein-antigen capturing module compound and preparing method and application thereof
  • Lipid binding protein-antigen capturing module compound and preparing method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0201] Embodiment 1: the expression of membrane protein on living cell (prokaryotic cell):

[0202] For the expression of membrane proteins on the surface of prokaryotic cells, Escherichia coli cells are used as an example to express TSPO (Translocator protein) membrane proteins on their cell membranes. According to literature reports, TSPO protein is expressed on the inner membrane of Escherichia coli, and its N-terminus is outside the membrane, while the C-terminus is inside the membrane (see image 3 ). Therefore, a His-tag (HHHHHH) is added to the N-terminus of the TSPO protein, and its final amino acid sequence is as follows:

[0203] HHHHHHNMDWALFLTFLAASGAPATTGALLKPDEWYDNLNKPWWNPPRWVFPLAWTSLYFLMSLAAMRVAQLEGSGQALAFYAAQLAFNTLWTPVFFGMKRMATALAVVMVMWLFVAATMWAFFQLDTWAGVLFVPYLIWATAATGLNFEAMRLN (SEQ ID NO. 6)

[0204] The His-TSPO gene was optimized to the preferred codon sequence of Escherichia coli, and after the sequence was artificially synthesized, it was cloned between t...

Embodiment 2

[0206] Example 2: Preparation of nanodisk "framework protein" MSP and Saposin

[0207] For the MSP protein, its wild-type and cysteine ​​mutants need to be prepared separately. See SEQ ID NO.5 for the full-length amino acid sequence of wild-type MSP, and see SEQ ID NO.1 and SEQ ID NO.2 for the amino acid sequences of MSP cysteine ​​mutants.

[0208] MGSSHHHHHHENLYFQGSTFSKLREQLGPVTQEFWDNLEKETEGLRQEM S KDLEEVK A KVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ (SEQ ID NO. 5)

[0209] SEQ ID NO.1 MSP cysteine ​​mutant S86C amino acid sequence (wherein, the mutated C is underlined and located at position 50)

[0210] MGSSHHHHHHENLYFQGSTFSKLREQLGPVTQEFWDNLEKETEGLRQEM C KDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ

[0211] SEQ ID NO.2 MSP cysteine ​​mutant A95C amino ac...

Embodiment 3

[0224] Embodiment 3: the preparation of affinity probe

[0225] Taking His-tag as an example for the affinity tag, the affinity probe can be a small molecule with high affinity to His-tag, such as tri-NTA, or a high-affinity protein macromolecule such as anti-His-tag scFv.

[0226] (1) Synthesis of Tri-NTA

[0227] For the synthetic route of Tri-NTA see Figure 5 .

[0228] First synthesize compound 1: tert-Butyl bromoacetate (tert-Butyl bromoacetate) (3ml, 20mmol) and EDIAP (4.3ml, 25mmol) were added in the DMF solution (40ml) of compound 0 (1.65g, 5mmol), under nitrogen Heated to 55°C under deoxygenation conditions and stirred continuously overnight. The reaction solution was heated to 60°C to remove volatile matter, and then 20ml of ethyl acetate was added and filtered. The precipitate was washed three more times with ethyl acetate. The filtrate was collected and concentrated. Purify with a silica gel column, and the mobile phase is cyclohexane / ethylacetate (3:1), to ...

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Abstract

The invention provides a lipid binding protein-antigen capturing module compound and a preparing method and application thereof, and particularly provides a compound with a structure of R1-(CM)m. An antigen capturing module CM can be located at any position of a lipid binding protein, R1 is the lipid binding protein, the CM is the antigen capturing module, is located at any position of the R1, andhas a structure of A1-R2-A2-R3, the A1 and A2 independently do not exist or are linking groups respectively, the R2 is a linker, the R3 is an affinity probe, and '-' represents bonds. The invention further provides a method for direct targeting extraction of membrane protein native antigens from cell membranes through the compound and the prepared native antigens.

Description

technical field [0001] The invention belongs to the field of biotechnology. Specifically, the present invention relates to a lipid-binding protein-antigen capture module complex, its preparation method and application. Background technique [0002] Antibody drugs refer to the general term for biological drugs derived from the specific recognition of a target by monoclonal antibodies, including monoclonal antibodies, bispecific antibodies, nanobodies and antibody Antibody-drug conjugate, etc. In recent years, antibody drugs have become the mainstream of biological drugs. [0003] Most antibody drugs target membrane proteins such as receptors on the cell membrane to inhibit ligand binding or activate intracellular pathways. Since the complete receptor contains a transmembrane domain (TMD), it is difficult to express in a soluble form in various expression systems (such as Escherichia coli, yeast, mammalian cells, etc.). [0004] At present, the commonly used ways to obtain...

Claims

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Application Information

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IPC IPC(8): C07K14/00C07K1/107G01N33/53C07K16/00
CPCC07K14/00C07K19/00G01N33/53C07K16/00
Inventor 周界文潘利强曹婵
Owner ASSEMBLY MEDICINE LLC
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