Method for detecting interaction between protein molecules based on label-gripper of magnetic tweezer system

Abstract

The invention relates to a label-gripper detection method for interaction between protein molecules based on a magnetic tweezer system. The method comprises the following steps: respectively carrying out fusion expression on a target protein and a label I, and carrying out fusion expression on a to-be-screened protein molecule and a label II; carrying out fusion expression on the connecting peptide, the gripper I and the gripper II; the gripper I specifically corresponds to the label I, and the gripper II specifically corresponds to the label II; putting any one of the obtained target protein and the to-be-screened protein molecules into a magnetic tweezer system sample pool, and performing incubation combination with a bottom plate of the sample pool; the other one is put into a sample pool after being combined with a magnetic ball in an incubation manner; finally, the connecting peptide is put into a sample pool, and a gripper I and a gripper II of the connecting peptide are respectively incubated and combined with the label I and the label II; a magnetic tweezer system is adopted, the distance between a magnetic ball and a bottom plate of a sample pool of the magnetic tweezer system is detected by gradually increasing the load force, and interaction between the target protein and the protein molecules to be screened is obtained. According to the invention, interaction between protein molecules can be intuitively reflected.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology, and particularly relates to a method for detecting protein - protein interactions based on a magnetic tweezer system using a tag - gripper approach. Background Art

[0002] With the development of life sciences, the demand for single - molecule research of biological macromolecules has been increasing, and single - molecule manipulation techniques such as optical tweezers and magnetic tweezers have been developed. Traditional methods for studying protein - protein interactions are mostly at the cell population or multi - molecule level and cannot directly observe the dynamic interactions of single molecules. The single - molecule magnetic tweezer technology has emerged, which can manipulate and measure proteins at the single - molecule level, providing a more accurate means for research in this field.

[0003] Magnetic tweezers exert a force on superparamagnetic beads using a magnetic field to affect the connected proteins. Its principle is unique and has obvious advantages. Compared with other single - molecule manipulation techniques, it has a large and wide - range force, is stable and lossless, can operate from the outside and can also penetrate into living cells to operate on fresh samples, can avoid damage to the sample activity caused by heat and light, has low requirements for samples, is resistant to system drift and noise, and can exert a force in the range of 0.1 pN to 100 pN.

[0004] The cross - integration of biotechnology with multiple disciplines has promoted the development of single - molecule magnetic tweezer technology and its combination with techniques such as single - molecule fluorescence. In recent years, this technology has achieved fruitful results in the field of protein - protein interactions, such as in protein folding kinetics, protein - nucleic acid interactions, etc. These results provide new insights for theoretical research and also provide potential strategies for disease diagnosis and treatment, such as discovering the influence of protein folding intermediate states and the real - time process of protein - nucleic acid interactions, which has strongly promoted the in - depth study of protein - protein interactions.

[0005] At present, the key targets of many diseases have been found, but the screening and development of agonist or inhibitor polypeptides corresponding to the targets are very slow. The main reason is that the current screening methods require the fusion expression of the target and the suspected target proteins before further research, and the suspected target protein library is very large, so the screening speed is restricted. Summary of the Invention

[0006] Aiming at the deficiencies of the existing technology, the present invention proposes a method for detecting protein - protein interactions based on a magnetic tweezer system using a tag - gripper approach. The specific technical solutions are as follows:

[0007] A method for detecting protein - protein interactions based on a magnetic tweezer system using a tag - gripper approach, comprising the following steps:

[0008] S1: First, fuse and express the target target protein with tag one to obtain the target target protein with tag one;

[0009] S2: Fuse and express the protein molecule to be screened with tag two to obtain the protein molecule to be screened with tag two;

[0010] S3: Fuse and express the linker peptide with catcher 1 and catcher 2 to obtain a linker peptide with catcher 1 at one end and catcher 2 at the other end; wherein, catcher 1 specifically corresponds to tag 1, and catcher 2 specifically corresponds to tag 2.

[0011] S4: Place either the target target protein with tag 1 obtained in S1 or the protein molecule to be screened with tag 2 obtained in S2 in the sample cell of the magnetic tweezer system and incubate and bind it to the bottom plate of the sample cell of the magnetic tweezer system; after incubating and binding the other one of the target target protein with tag 1 obtained in S1 and the protein molecule to be screened with tag 2 obtained in S2 to magnetic beads, place it in the sample cell of the magnetic tweezer system.

[0012] Place the linker peptide with catcher 1 and catcher 2 obtained in S3 in the sample cell of the magnetic tweezer system, and the catcher 1 and catcher 2 of the linker peptide are respectively incubated and bound to tag 1 on the target target protein obtained in S1 and tag 2 on the protein molecule to be screened obtained in S2.

[0013] S5: Use the magnetic tweezer system to detect the distance between the magnetic beads and the bottom plate of the sample cell of the magnetic tweezer system by gradually increasing the loading force, and compare it with the length of the linker peptide:

[0014] If the distance between the magnetic beads and the bottom plate of the sample cell reaches the length of the linker peptide before the applied loading force reaches the set threshold, it is considered that there is no interaction between the target target protein and the protein molecule to be screened; otherwise, it is considered that there is an interaction between the target target protein and the protein molecule to be screened; and, the more the applied loading force exceeds the set threshold, the stronger the interaction between the target target protein and the protein molecule to be screened is considered.

[0015] Further, in step S5, the loading force at which there is no interaction between the target target protein and the protein molecule to be screened and the length of the linker peptide is reached is used as the set threshold.

[0016] Further, the linker peptide does not interact with the target target protein.

[0017] The beneficial effects of the present invention are as follows:

[0018] (1) The present invention creatively introduces the tag-catcher that can specifically bind to each other without interference into the magnetic tweezer system. Each tag-catcher can capture a target protein molecule. Through multiple groups of tag-catchers, it can capture multiple target protein molecules simultaneously, so as to study the interaction between the captured protein molecules by using the single-molecule magnetic tweezer technology.

[0019] (2) The method of the present invention does not require the fusion expression of the target and the suspected target proteins one by one. Instead, they are expressed separately. Only a specific tag needs to be added during the expression, and then it can bind to the tandem catcher together with the target. The interaction between the target and the target protein in a certain space is measured, collected, and analyzed using a single-molecule magnetic tweezer. The present invention can intuitively reflect the interaction between protein molecules; and the amount of protein required is less. Only by replacing different suspected target proteins can the screening work be completed. The present invention can be used for the screening of macromolecular drugs for known targets of related diseases such as cancer, and can facilitate the rapid and efficient detection and screening of magnetic tweezers. Description of the Drawings

[0020] Figure 1 It is a schematic diagram of the tags and the corresponding catchers in this embodiment. Among them, Fig. (a1) is Tag 1, (a2) is Tag 2, (a3) is Tag 3, (b1) is Catcher 1, (b2) is Catcher 2, and (b3) is Catcher 3.

[0021] Figure 2 It is a schematic diagram of the fusion expression of the target target protein and Tag 1, the protein molecule to be screened and Tag 2, and the fusion expression of the linker peptide with Catcher 1 and Catcher 2. Among them, Fig. (a) is a schematic diagram of the fusion expression of the target target protein and Tag 1; Fig. (b) is a schematic diagram of the fusion expression of the protein molecule to be screened and Tag 2, and Fig. (c) is a schematic diagram of the fusion expression of the linker peptide with Catcher 1 and Catcher 2.

[0022] Figure 3 It is a schematic diagram of using a magnetic tweezer system to detect the distance between the magnetic bead and the bottom plate of the sample cell of the magnetic tweezer system. Detailed Embodiments

[0023] The present invention will be described in detail below according to the drawings and preferred embodiments. The purpose and effect of the present invention will become more apparent. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0024] As Figure 1 shown, three examples of the tag-catcher combinations involved in the method of the present invention are given. The arc surfaces, triangles, polygons, etc. between the tags and the corresponding catchers have no specific meaning, but are only used to indicate the specificity between the two.

[0025] As Figure 2 and Figure 3 shown, the method for detecting the interaction between proteins of the present invention includes the following steps:

[0026] S1: First, the target target protein and Tag 1 are fused and expressed to obtain the target target protein with Tag 1. As Figure 2as shown in (a) therein;

[0027] S2: Co - express the protein molecule to be screened and tag two respectively to obtain the protein molecule to be screened with tag two, as Figure 2 shown in (b) therein;

[0028] S3: Co - express the linker peptide with catcher one and catcher two to obtain a linker peptide with catcher one at one end and catcher two at the other end; wherein, catcher one specifically corresponds to tag one, and catcher two specifically corresponds to tag two; as Figure 2 shown in (c) therein;

[0029] S4: Place any one of the target target protein with tag one obtained in S1 and the protein molecule to be screened with tag two obtained in S2 in the sample pool of the magnetic tweezer system and incubate and bind it to the bottom plate of the sample pool of the magnetic tweezer system; after incubating and binding the other of the target target protein with tag one obtained in S1 and the protein molecule to be screened with tag two obtained in S2 to magnetic beads, place it in the sample pool of the magnetic tweezer system;

[0030] Place the linker peptide with catcher one and catcher two obtained in S3 in the sample pool of the magnetic tweezer system, and the catcher one and catcher two of the linker peptide respectively incubate and bind to tag one on the target target protein obtained in S1 and tag two on the protein molecule to be screened obtained in S2; as Figure 3 shown.

[0031] S5: Use the magnetic tweezer system to detect the distance between the magnetic beads and the bottom plate of the sample pool of the magnetic tweezer system by gradually increasing the loading force, and compare it with the length of the linker peptide:

[0032] If the distance between the magnetic beads and the bottom plate of the sample pool reaches the length of the linker peptide before the applied loading force reaches the set threshold, it is considered that there is no interaction between the target target protein and the protein molecule to be screened; otherwise, it is considered that there is an interaction between the target target protein and the protein molecule to be screened; and, the more the applied loading force exceeds the set threshold, the stronger the interaction between the target target protein and the protein molecule to be screened is considered.

[0033] In order to study the interaction between multiple target protein molecules, two sets or multiple sets of tag - catcher can also be used to detect by using the method of the present invention. The flexibility of different sets of tag - catcher and the linker peptide between the two catchers can be different.

[0034] By using the method of the present invention, only by replacing different suspected target proteins, the screening work can be completed, which greatly improves the screening efficiency of agonist or inhibitory polypeptides corresponding to the target.

[0035] Those of ordinary skill in the art can understand that the above are only preferred examples of the invention and are not used to limit the invention. Although the invention has been described in detail with reference to the foregoing examples, for those skilled in the art, they can still modify the technical solutions described in the foregoing examples or make equivalent replacements for some of the technical features. Any modifications, equivalent replacements, etc. made within the spirit and principle of the invention shall be included within the protection scope of the invention.

Claims

1. A method for detecting the interaction between protein molecules using a label-gripper based on a magnetic tweezers system, characterized in that: The steps include: S1: First, the target protein and tag one are fused and expressed to obtain the target protein with tag one; S2: expressing the protein molecule to be screened by fusion with label 2 to obtain the protein molecule to be screened with label 2; S3: The connecting peptide is fused with gripper 1 and gripper 2 to obtain a connecting peptide with gripper 1 at one end and gripper 2 at the other end; wherein gripper 1 specifically corresponds to label 1, and gripper 2 specifically corresponds to label 2; S4: placing any one of the target protein with label one obtained by S1 and the protein molecule to be screened with label two obtained by S2 in the sample pool of the magnetic tweezers system, and incubating and combining with the bottom plate of the sample pool of the magnetic tweezers system; incubating and combining the other one of the target protein with label one obtained by S1 and the protein molecule to be screened with label two obtained by S2 with the magnetic ball, and then placing them in the sample pool of the magnetic tweezers system; The connecting peptide with gripper 1 and gripper 2 obtained by S3 is placed in the sample pool of the magnetic tweezers system, and the gripper 1 and gripper 2 of the connecting peptide are incubated and combined with the label 1 on the target protein obtained by S1 and the label 2 on the protein molecule to be screened obtained by S2 respectively; S5: Using the magnetic tweezers system, by gradually increasing the load force, the distance between the magnetic ball and the bottom plate of the sample pool of the magnetic tweezers system is detected and compared with the length of the connecting peptide: If the distance between the magnetic ball and the bottom plate of the sample pool reaches the length of the connecting peptide before the applied load force reaches the set threshold, it is considered that there is no interaction between the target protein and the protein molecule to be screened; otherwise, it is considered that there is interaction between the target protein and the protein molecule to be screened; and, the more the applied load force exceeds the set threshold, the stronger the interaction between the target protein and the protein molecule to be screened is considered.

2. The method for detecting the interaction between protein molecules by using a label-gripper based on a magnetic tweezers system according to claim 1, characterized in that: In the step S5, the loading capacity at which the length of the connecting peptide is reached without any interaction between the target protein and the protein molecule to be screened is set as a threshold.

3. The method for detecting the interaction between protein molecules by using a label-gripper based on a magnetic tweezers system according to claim 1, characterized in that: The connecting peptide does not interact with the target protein.

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