CN-122011083-A - Reversible affinity purification system, method and application based on host-guest interaction

CN122011083ACN 122011083 ACN122011083 ACN 122011083ACN-122011083-A

Abstract

The invention discloses a reversible affinity purification system based on host-guest interaction, a method and application thereof, wherein the system comprises a functionalized solid-phase carrier, a main molecule is covalently modified on the surface of the functionalized solid-phase carrier, a bait molecule is connected with a guest molecule, wherein the guest molecule and the main molecule can interact with each other in a specific and reversible manner, so that the bait molecule is fixed on the functionalized solid-phase carrier, and the combination of the guest molecule and the main molecule can be reversibly dissociated by adding a free competitive guest molecule, so that the elution of the bait molecule or a compound thereof from the functionalized solid-phase carrier is realized. The invention has the dynamic reversible regulation and control characteristics, can obtain high-activity target protein through mild competitive elution, can effectively avoid the interference of endogenous biotin, and has the advantages of repeated utilization of the functionalized solid phase carrier and stronger environmental suitability for complex biological samples.

Inventors

XIAO LIN
GAO YANMEI
CHEN YUCHI
GUO XIAOLAN
LV JIAZHEN
HE HONGFEI

Assignees

川北医学院

Dates

Publication Date: 20260512
Application Date: 20260210

Claims (10)

1. A reversible affinity purification system based on host-guest interactions, comprising: (a) A functionalized solid phase carrier, the surface of which is covalently modified with a host molecule; (b) A bait molecule having a guest molecule attached thereto; Wherein the guest molecule is capable of specific, reversible host-guest interactions with the host molecule to immobilize the bait molecule to the functionalized solid phase carrier, and wherein binding of the guest molecule to the host molecule can be reversibly dissociated by addition of a free competing guest molecule to effect elution of the bait molecule or complex thereof from the functionalized solid phase carrier.
2. The reversible affinity purification system based on the action of a host and a guest according to claim 1, wherein the host molecule is cucurbituril [7] uril and the guest molecule comprises ferrocene, diadamantane, bicyclo [2.2.2] octane, 1, 4-diazabicyclo [2.2.2] octane or an adamantane derivative.
3. The reversible affinity purification system based on host-guest interaction according to claim 1 or 2, wherein the functionalized solid phase carrier is prepared by covalently attaching azide-modified cucurbituril [7] urils to the surface of an alkynyl-modified solid phase matrix by click chemistry.
4. The host-guest interaction-based reversible affinity purification system according to claim 1, wherein the decoy molecule is a small molecule compound, a protein, a polypeptide, an antibody or a nucleic acid.
5. The reversible affinity purification system based on the action of a host and a guest according to claim 1, wherein the solid phase matrix in the functionalized solid phase carrier is a magnetic bead.
6. A method of reversible affinity purification of biomolecules for isolating target molecules interacting with bait molecules, characterized in that the method uses a reversible affinity purification system according to any one of claims 1 to 5 and comprises the steps of: (1) Fixing the bait molecules connected with the guest molecules on a functionalized solid-phase carrier with the surface modified with the host molecules through interaction of the host and the guest; (2) Contacting the immobilized decoy molecule with a biological sample containing potentially interacting target molecules; (3) Separating and washing the functionalized solid support; (4) Eluting the decoy molecule-target molecule complex from the functionalized solid support by adding a free competing guest molecule, thereby obtaining a purified target molecule.
7. The reversible affinity purification method according to claim 6, wherein in step (4), the free competitive guest molecule is an amantadine compound at a concentration of 0.1mM to 10mM in the eluent.
8. A kit comprising the reversible affinity purification system of any one of claims 1 to 5.
9. The kit of claim 8, further comprising a free competing guest molecule.
10. Use of the reversible affinity purification system of any one of claims 1 to 5, or the reversible affinity purification method of any one of claims 6 to 7, or the kit of any one of claims 8 to 9 in a biomolecular interaction study.

Description

Reversible affinity purification system, method and application based on host-guest interaction Technical Field The invention relates to the technical field of biology, in particular to a reversible affinity purification system based on the action of a host and a guest, a method and application thereof. Background In the life science research and drug development process, the clear interaction among biomolecules and the accurate separation of target proteins are core preconditions for analyzing the vital activity mechanism and discovering potential drug targets. Currently, pull-Down technology is the dominant means to achieve the above objective. The core principle is that the target protein is interacted by the capture of the marked bait molecule, and then the identification is carried out after separation and purification. The technology has the advantages of being direct and efficient, is widely applied to screening and verification of various biological molecular interaction relations such as protein-small molecular compounds, protein-protein, protein-nucleic acid and the like, and is a key technical support for analyzing signal paths and functional complex compositions in basic scientific research, discovering target proteins in drug research and development and verifying drug-target interaction. The prior art is dependent on the biotin-streptavidin system. The binding of streptavidin to biotin is a highly optimized molecular "key" mechanism that relies on various intermolecular forces such as hydrogen bonding, hydrophobic interactions, and van der waals forces to form extremely stable complexes. The core principle of the system is to realize the fixation of the bait and the capture separation of the target protein by utilizing the non-covalent interaction (the binding constant Kd is about 10 -14 M) with ultrahigh affinity between biotin and streptavidin. The core implementation steps comprise 1, labeling biotin on a bait by adopting a chemical crosslinking method, 2, incubating a biotin-labeled bait molecule and a streptavidin-modified solid-phase carrier (such as magnetic beads and agarose gel) to enable the bait molecule to be fixed on the surface of the carrier through the action of biotin-streptavidin, 3, adding a sample (such as cell lysate) containing potential target proteins, incubating to enable the bait molecule and the target proteins to form a complex, 4, washing for multiple times to remove the impurity proteins, and eluting the target proteins through strong denaturation or extreme pH conditions to finally obtain the purified target proteins for subsequent identification. The system has the advantages of high binding specificity, strong stability and mature technology, and is the mainstream technology of the current biological macromolecule interaction research. However, despite the wide application of this system, in practice it still presents a significant technical bottleneck: The elution condition is harsh, the target protein is easy to be inactivated, and when the combination of biotin and streptavidin is almost irreversible, and the captured target protein complex is eluted later, the harsh conditions such as strong denaturant, extreme pH or high temperature are needed. These conditions are extremely prone to denaturation, aggregation or inactivation of the target protein, severely affecting the likelihood that it will be subsequently used in functional studies such as structural resolution, enzyme activity assays, etc. Endogenous interference results in false positives-endogenous biotinylated proteins are naturally present in many biological samples (e.g., cells, tissues, serum). These endogenous biotins bind non-specifically to the streptavidin solid phase carriers in the system, producing high background noise and false positive signals, severely interfering with the accuracy and reliability of experimental results. The carrier is not reusable, the cost is high, and because of the irreversibility of the combination of biotin-streptavidin, the solid phase carrier (such as magnetic beads and agarose beads) is invalid after one capturing-eluting cycle, the regeneration and the recycling can not be realized, and the experimental cost is increased. The suitability of the system is limited, the activity of the streptavidin is greatly influenced by conditions such as environmental pH, ionic strength and the like, and in biological samples with complex components (such as cell lysate and tissue homogenate), the combination efficiency and stability of the streptavidin can be reduced, so that the application of the system under wider or harsher physiological conditions is limited. Therefore, providing a novel pull-Down technology that can achieve mild elution to maintain target protein activity, avoid endogenous substance interference, and reuse solid phase carriers is a technical problem to be solved in the art. Disclosure of Invention In order to solve the technical problems, the invent