Protein coupling method, protein complex and application

Abstract

The invention provides a protein coupling method, a protein complex and application. The method comprises the following steps: a first protein and a second protein are subjected to coupling treatment, the first protein is connected with DBCOTAG, and the second protein is connected with DBCO. Furthermore, the method can realize a coupling mode determined by the luciferase and the antibody half site, so that the stability of a coupling product is improved, and the activity of the coupling product is prevented from being reduced.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and specifically, to a protein coupling method, a complex and uses, and in particular to a method for coupling two proteins, a protein complex, a molecular detection method, a method for determining the location of a target molecule and a kit. Background Art

[0002] Bioconjugation is a process in which one molecule is attached to another molecule through a covalent bond to produce a complex consisting of two molecules connected together. Bioconjugation reactions are usually carried out under the mediation of chemical coupling agents or enzymes. In industrial applications, chemical coupling agents are often used for coupling reactions. However, chemical coupling greatly affects the activity of the coupling product.

[0003] Therefore, there is an urgent need in the art for a new coupling method to ensure that the activity of the coupling product is not affected. Summary of the invention

[0004] This application is filed by the inventor based on the discovery of the following facts and problems:

[0005] SMCC (succinimidyl 4-(N-maleimidyl) cyclohexane-1-carboxylate) coupling is a technique that uses SMCC coupling agents to cross-link a protein containing a primary amine and a sulfhydryl group. One end of the SMCC coupling agent is N-hydroxysuccinimide ester (NHS esters, NHS ester), and the other end is maleimide. Because the maleimide group in the SMCC molecule needs to react with the free sulfhydryl group in the protein, it will destroy the natural structure of the protein, resulting in a decrease in the natural activity of the protein or even inactivation. Because the experimental process is linear, the HNS ester must first react with one protein (such as luciferase) and then react with another protein (such as digoxin antibody (Ab-Dig)), which greatly reduces the flexibility of experimental design during the development process.

[0006] In view of this, the present application proposes a method for solving the problem of reduced activity of coupled products during protein coupling.

[0007] In the first aspect of the present application, the present application proposes a method for coupling two proteins. According to an embodiment of the present application, the method comprises: coupling a first protein with a second protein, wherein the first protein is connected to a DBCOTAG (dibenzylcyclooctyne tag), and the second protein is connected to a DBCO (dibenzylcyclooctyne).

[0008] According to the embodiments of the present application, the protein coupling method based on the specific nucleophilic addition reaction of DBCOTAG and DBCO has higher product stability and activity level than the current thiol coupling method.

[0009] In some examples, the so-called DBCOTAG has an amino acid sequence as shown in SEQ ID NO:1.

[0010] LCYPWVY (SEQ ID NO: 1).

[0011] In some examples, the DBCO has a structure as shown in formula (I):

[0012]

[0013] According to an embodiment of the present application, the protein coupling method may further include at least one of the following technical features:

[0014] According to an embodiment of the present application, the second protein is connected to DBCO via an amide bond or coupled via a thiol group. In some preferred embodiments of the present application, the second protein is connected to DBCO via an amide bond.

[0015] According to an embodiment of the present application, the second protein is connected to DBCO through an amide bond by the following method: the second protein is contacted with DBCO connected with N-hydroxysuccinimide (NHS esters), and the second protein has free -NH2. The so-called contact treatment includes: under weak alkaline conditions, since the second protein has free -NH2 and DBCO has NHS ester modification, the free -NH2 undergoes an amidation reaction (Amide Formation Reaction) with the NHS ester to produce a stable amide bond.

[0016] According to an embodiment of the present application, in the contact system, the molar ratio of the second protein to be modified to DBCO linked to N-hydroxysuccinimide is 1:5 to 1:20. In some examples, the molar ratio of the second protein to be modified to DBCO linked to N-hydroxysuccinimide is optionally 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19 or 1:20.

[0017] According to an embodiment of the present application, before the coupling treatment, the first protein is further subjected to a reduction treatment. Since DBCOTAG contains sulfhydryl groups, it is easy to form intermolecular disulfide bonds, and the reactive sulfhydryl groups are exposed after the reduction treatment.

[0018] According to an embodiment of the present application, the reduction treatment is performed in the presence of a reducing agent, which can reduce intermolecular disulfide bonds to expose reactive sulfhydryl groups in proteins.

[0019] According to an embodiment of the present application, the reducing agent includes at least one selected from DTT (dithiothioate), β-mercaptoethanol and TCEP (tris-(2-carboxyethyl)phosphine hydrochloride). In some examples, the reducing agent includes but is not limited to the above-mentioned ones, and methyl mercaptoethane sulfonate (MESNA) or ornithine disulfide (NAC) can also be selected.

[0020] According to an embodiment of the present application, in the coupling treatment system, the molar ratio of the first protein to the second protein is 5: 1 to 1: 1. In some examples, the molar ratio of the first protein to the second protein can be 5: 1, 4: 1, 3: 1, 2: 1 or 1: 1.

[0021] According to an embodiment of the present application, the DBCO linked to N-hydroxysuccinimide is provided in the form of DBCO-(PEG)n-NHS, wherein n is an integer between 0 and 12; in some examples of the present application, n is preferably 0, 4, 8 or 12.

[0022] In some examples of the present application, the structural formula of DBCO-(PEG)n-NHS is shown in Formula (II) to Formula (III).

[0023]

[0024]

[0025] According to an embodiment of the present application, the N-terminus or C-terminus of the first protein is connected to DBCOTAG.

[0026] According to an embodiment of the present application, the C-terminus of the first protein is connected to the N-terminus of the DBCOTAG. In some specific examples, the inventor unexpectedly found that the C-terminus of the first protein is connected to the N-terminus of the DBCOTAG, and the coupling efficiency and expression efficiency are higher.

[0027] According to an embodiment of the present application, the N-terminus or C-terminus of the first protein is connected to DBCOTAG via a connecting peptide. In some examples of the present application, adding a connecting peptide can increase the stability and solubility of the protein and avoid folding or aggregation during protein expression.

[0028] According to an embodiment of the present application, the connecting peptide includes at least one selected from a flexible peptide and a rigid peptide.

[0029] According to an embodiment of the present application, the amino acid sequence of the flexible peptide is (GGGGS) n1 ; wherein n1 is an integer greater than or equal to 1, preferably 1, 2 or 3.

[0030] According to an embodiment of the present application, the amino acid sequence of the rigid peptide is (EAAAK) n2; wherein n2 is an integer greater than or equal to 1, preferably 1, 2 or 3.

[0031] According to an embodiment of the present application, the N-terminus or C-terminus of the first protein is further connected with a His tag. In some preferred embodiments of the present application, the His tag is 6*His. In some examples, the amino acid sequence of the 6*His tag is: HHHHHH.

[0032] According to an embodiment of the present application, the N-terminus of the first protein is connected to a His tag, the C-terminus of the DBCOTAG is connected to the N-terminus of the His tag, or the N-terminus of the DBCOTAG is connected to the C-terminus of the first protein. Specifically, there are two connection modes: DBCOTAG-His tag-first protein or His tag-first protein-DBCOTAG.

[0033] According to an embodiment of the present application, the N-terminus of the DBCOTAG is connected to the C-terminus of the first protein. The inventors found that the connection between the N-terminus of the DBCOTAG and the C-terminus of the first protein has a higher coupling efficiency.

[0034] According to an embodiment of the present application, the C-terminus of the first protein is connected to a His tag, the C-terminus of the DBCOTAG is connected to the N-terminus of the first protein, or the N-terminus of the DBCOTAG is connected to the C-terminus of the His tag. Specifically, there are two connection modes: DBCOTAG-first protein-His tag or first protein-His tag-DBCOTAG.

[0035] According to an embodiment of the present application, the N-terminus of the DBCOTAG is connected to the C-terminus of the His tag.

[0036] According to an embodiment of the present application, the C-terminus of the DBCOTAG is connected to the N-terminus of the His tag, the C-terminus of the His tag is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the first protein; or the C-terminus of the His tag is connected to the N-terminus of the first protein, the C-terminus of the first protein is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the DBCOTAG. Specifically, there are two connection modes: DBCOTAG-His tag-connecting peptide-first protein or His tag-first protein-connecting peptide-DBCOTAG.

[0037] According to an embodiment of the present application, the C-terminus of the DBCOTAG is connected to the N-terminus of the first protein, the C-terminus of the first protein is connected to the N-terminus of the connecting peptide, the C-terminus of the connecting peptide is connected to the N-terminus of the His tag; or the C-terminus of the first protein is connected to the N-terminus of the connecting peptide, the C-terminus of the connecting peptide is connected to the N-terminus of the His tag, and the C-terminus of the His tag is connected to the N-terminus of the DBCOTAG. Specifically, there are two connection modes: DBCOTAG-first protein-connecting peptide-His tag or first protein-connecting peptide-His tag-DBCOTAG.

[0038] According to an embodiment of the present application, the first protein and the second protein are independently selected from luciferase and antibodies, respectively.

[0039] In some examples of the present application, the first protein is selected from luciferase, and the second protein is selected from antibodies.

[0040] In some other examples of the present application, the first protein is selected from antibodies, and the second protein is selected from luciferase.

[0041] According to an embodiment of the present application, the luciferase includes at least one selected from Gaussia luciferase or mutant, Nano luciferase or mutant, bacterial luciferase or mutant, firefly luciferase or mutant, sea fluorescein luciferase or mutant, Renilla luciferase or mutant, and spiny shrimp luciferase or mutant.

[0042] According to an embodiment of the present application, the luciferase is Gaussia luciferase.

[0043] According to an embodiment of the present application, the antibody is selected from at least one of a digoxin antibody, an A base antibody, a G base antibody, a C base antibody, a U base antibody or a T base antibody.

[0044] According to an embodiment of the present application, the antibody is selected from digoxin antibody.

[0045] In the second aspect of the present application, the present application proposes a protein complex. According to an embodiment of the present application, the protein complex is prepared by the method described in the first aspect of the present application. In some examples of the present application, the protein complex can be applied to the field of molecular detection or the field of fluorescent tracing, has low cost, and can be widely used.

[0046] In the third aspect of the present application, the present application proposes a protein complex. According to an embodiment of the present application, the protein complex comprises: a first protein, the first protein is connected to DBCOTAG, preferably, the N-terminus or C-terminus of the first protein is connected to DBCOTAG; and a second protein, the second protein is connected to DBCO, and the DBCOTAG is connected to the DBCO through a carbon-sulfur bond. In some examples of the present application, the protein complex can be applied to the field of molecular detection or the field of fluorescent tracing, has low cost, and can be widely used.

[0047] According to an embodiment of the present application, the above protein complex further includes at least one of the following technical features:

[0048] According to an embodiment of the present application, the second protein is connected to DBCO via an amide bond or coupled via a thiol group. In some preferred embodiments of the present application, the second protein is connected to DBCO via an amide bond.

[0049] According to an embodiment of the present application, the second protein is connected to DBCO through an amide bond by the following method: the second protein is contacted with DBCO connected with N-hydroxysuccinimide (NHS esters), and the second protein has free -NH2. The so-called contact treatment includes: under weak alkaline conditions, since the second protein has free -NH2 and DBCO has NHS ester modification, the free -NH2 reacts with the NHS ester to form an amidation reaction (Amide Formation Reaction) to produce a stable amide bond.

[0050] According to an embodiment of the present application, the first protein and the second protein are independently selected from luciferase and antibodies, respectively.

[0051] In some examples of the present application, the luciferase includes at least one selected from Gaussia luciferase or mutant, Nano luciferase or mutant, bacterial luciferase or mutant, firefly luciferase or mutant, sea fluorescein luciferase or mutant, sea renilla luciferase or mutant, and shrimp luciferase or mutant. In some preferred examples of the present application, the luciferase is Gaussia luciferase.

[0052] In some examples of the present application, the antibody is selected from at least one of digoxin antibody, A base antibody, G base antibody, C base antibody, U base antibody or T base antibody. In some preferred examples of the present application, the antibody is selected from digoxin antibody.

[0053] According to an embodiment of the present application, the N-terminus or C-terminus of the first protein is connected to DBCOTAG via a connecting peptide. In some examples of the present application, adding a connecting peptide can increase the stability and solubility of the protein and avoid folding or aggregation during protein expression.

[0054] According to an embodiment of the present application, the connecting peptide includes at least one selected from a flexible peptide and a rigid peptide.

[0055] According to an embodiment of the present application, the amino acid sequence of the flexible peptide is (GGGGS) n1 ; wherein n1 is an integer greater than or equal to 1, preferably 1, 2 or 3.

[0056] According to an embodiment of the present application, the amino acid sequence of the rigid peptide is (EAAAK) n2 ; wherein n2 is an integer greater than or equal to 1, preferably 1, 2 or 3.

[0057] According to an embodiment of the present application, the N-terminus or C-terminus of the first protein is further connected with a His tag. In some preferred embodiments of the present application, the His tag is 6*His. In some examples, the amino acid sequence of the 6*His tag is: HHHHHH.

[0058] According to an embodiment of the present application, the N-terminus of the first protein is connected to a His tag, the C-terminus of the DBCOTAG is connected to the N-terminus of the His tag, or the N-terminus of the DBCOTAG is connected to the C-terminus of the first protein. Specifically, there are two connection modes: DBCOTAG-His tag-first protein or His tag-first protein-DBCOTAG.

[0059] According to an embodiment of the present application, the N-terminus of the DBCOTAG is connected to the C-terminus of the first protein. The inventors found that the connection between the N-terminus of the DBCOTAG and the C-terminus of the first protein has a higher coupling efficiency.

[0060] According to an embodiment of the present application, the C-terminus of the first protein is connected to a His tag, the C-terminus of the DBCOTAG is connected to the N-terminus of the first protein, or the N-terminus of the DBCOTAG is connected to the C-terminus of the His tag. Specifically, there are two connection modes: DBCOTAG-first protein-His tag or first protein-His tag-DBCOTAG.

[0061] According to an embodiment of the present application, the N-terminus of the DBCOTAG is connected to the C-terminus of the His tag.

[0062] According to an embodiment of the present application, the C-terminus of the DBCOTAG is connected to the N-terminus of the His tag, the C-terminus of the His tag is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the first protein; or the C-terminus of the His tag is connected to the N-terminus of the first protein, the C-terminus of the first protein is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the DBCOTAG. Specifically, there are two connection modes: DBCOTAG-His tag-connecting peptide-first protein or His tag-first protein-connecting peptide-DBCOTAG.

[0063] According to an embodiment of the present application, the C-terminus of the DBCOTAG is connected to the N-terminus of the first protein, the C-terminus of the first protein is connected to the N-terminus of the connecting peptide, the C-terminus of the connecting peptide is connected to the N-terminus of the His tag; or the C-terminus of the first protein is connected to the N-terminus of the connecting peptide, the C-terminus of the connecting peptide is connected to the N-terminus of the His tag, and the C-terminus of the His tag is connected to the N-terminus of the DBCOTAG. Specifically, there are two connection modes: DBCOTAG-first protein-connecting peptide-His tag or first protein-connecting peptide-His tag-DBCOTAG.

[0064] In the fourth aspect of the present application, the present application proposes a molecular detection method. According to an embodiment of the present application, the method comprises: contacting the molecule to be detected with the protein complex described in the second aspect or the third aspect of the present application, wherein the first protein and the second protein are independently selected from luciferase and antibody, respectively; based on the signal after the contact treatment, determining the molecule; wherein the molecule to be detected carries the luciferase substrate or has binding activity with the antibody. According to an embodiment of the present application, the method is used to detect molecules (such as antigens or proteins, etc.) with high selectivity and high sensitivity. It has broad application prospects in biomedical research, diagnosis, drug screening and biotechnology.

[0065] According to an embodiment of the present application, the above molecular detection method may also include at least one of the following technical features:

[0066] According to an embodiment of the present application, the signal is a fluorescent signal. In some examples, the content or type of the molecule to be detected can be determined based on the strength or type of the fluorescent signal.

[0067] Exemplarily, a molecule to be tested containing a luciferase substrate is contacted with the protein complex provided in the present application. When the substrate binds to the luciferase, a fluorescent signal is generated. The fluorescent signal is detected by direct observation or with the aid of an instrument to determine the content or type of the molecule to be tested.

[0068] In the fifth aspect of the present application, the present application proposes a method for determining the position of a target molecule. According to an embodiment of the present application, the method comprises: contacting the target molecule with the protein complex described in the second aspect or the third aspect of the present application, wherein the first protein and the second protein are independently selected from luciferase and an antibody; based on the signal after the contact treatment, determining the position of the target molecule; wherein the target molecule carries a luciferase substrate or has binding activity with an antibody.

[0069] According to an embodiment of the present application, the method for determining the position of a target molecule may further include at least one of the following technical features:

[0070] According to an embodiment of the present application, the signal is a fluorescent signal. In some examples, the position of the target molecule can be determined based on the strength or type of the fluorescent signal.

[0071] In the sixth aspect of the present application, the present application proposes a kit. According to an embodiment of the present application, the kit includes the protein complex described in the second aspect or the third aspect of the present application. According to an embodiment of the present application, the kit can quickly detect the content, type or location of the molecule to be tested, and has the advantages of low cost, simple operation and portability.

[0072] According to an embodiment of the present application, the above-mentioned kit further includes at least one of the following technical features:

[0073] According to an embodiment of the present application, the kit further includes at least one of a luciferase substrate and a luciferase buffer.

[0074] According to specific embodiments of the present application, the protein complex and kit described in the present application can also be used for antigen detection.

[0075] It should be understood that within the scope of this application, the above-mentioned technical features of this application and the technical features specifically described below (such as implementation schemes) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the following description of the embodiments in conjunction with the accompanying drawings, in which:

[0077] Figure 1 This is a schematic diagram of the SDS-PAGE results of Gluc-DBCOTAG described in one embodiment of the present application;

[0078] Figure 2Schematic diagram of the screening results of the Gluc dimer experimental conditions described in one embodiment of the present application; wherein, A) DTT-reduced Gluc dimer SDS-PAGE; B) enzyme activity detection after dimer reduction; C) enzyme activity detection after monomer reduction;

[0079] Figure 3 This is a schematic diagram of the screening results of the DBCO and antibody ratio described in one embodiment of the present application;

[0080] Figure 4 This is a schematic diagram of the effect of different proportions of DBCO on the enzyme activity of the coupling product described in an embodiment of the present application;

[0081] Figure 5 This is a schematic diagram of the SDS-PAGE results of Gluc-AbDIG before purification described in an example of the present application;

[0082] Figure 6 This is a schematic diagram of the SDS-PAGE results of Gluc-AbDIG after purification described in an example of the present application;

[0083] Figure 7 This is a schematic diagram of the chemiluminescence detection results of the coupling product described in one embodiment of the present application;

[0084] Figure 8 It is a schematic diagram of the analysis results of the enzyme activity detection of the coupling product described in one embodiment of the present application. DETAILED DESCRIPTION

[0085] The embodiments of the present application are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present application, and cannot be understood as limiting the present application.

[0086] definition

[0087] In the description of the present application, it should be understood that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of the present application, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0088] In this article, "luciferase" belongs to a class of enzymes that catalyze light-emitting chemical reactions. The substrate of this enzyme is called "luciferin". In the absence of ATP, luciferin can be catalyzed by luciferase to produce a chemical reaction, which leads to light emission. Gaussia luciferase (Gaussia princeps luciferase, referred to as Gluc) is a luciferase isolated from the marine copepod Gaussia princeps, with a molecular weight of about 19.9kDa and composed of 185 amino acids. Gaussia luciferase can catalyze the enzymatic oxidation reaction of luciferin substrates (such as coelenterazine and its derivatives) and produce fluorescence with a wavelength between 460-480nm (the wavelength varies depending on the substrate).

[0089] Unlike the firefly luciferase system, Gaussia luciferase does not require the participation of high-energy molecules such as ATP and coenzyme A. The reaction only requires the participation of coelenterazine and oxygen, which greatly simplifies its use in a variety of application fields. This article uses Gaussia luciferase (Gaussia princeps luciferase, Gluc) as the first protein to exemplify the feasibility of the technical solution of this application.

[0090] Among them, the amino acid sequence of Gluc is shown in SEQ ID NO:2.

[0091] MKPTENNEDFNIVAVASNFATTDLDADRGKLPGKKLPLEVLKEMEANARKAGCTRGCLICLSHIKC TPKMKKFIPGRCHTYEGDKESAQGGIGEAIVDIPEIPGFKDLEPMEQFIAQVDLCVDCTTGCLKGLANVQ CSDLLKKWLPQRCATFASKIQGQVDKIKGAGGD (SEQ ID NO: 2).

[0092] In this article, IgG antibody (Immunoglobulin G) is a type of mammalian antibody, also known as immunoglobulin (Ig), with a molecular weight of about 150kDa. This application uses Antibody of Digoxin (Ab-Dig) as the second protein to exemplify the feasibility of the technical solution of this application.

[0093] The so-called second protein has free -NH2, i.e., primary amine (-NH2). Primary amines are present at the N-terminus of each polypeptide chain (called α-amino) and in the side chain of lysine (Lys, K) residues (called ε-amino). Since primary amines are positively charged under physiological conditions, they are usually outward (i.e., on the outer surface of the protein), making them easier to couple without changing the protein structure. However, in the coupling process with luciferase, the coupling reaction based on the reaction with primary amines is usually accompanied by the irreversible loss of luciferase enzyme activity. To this end, the inventors of the present application have found that DBCOTAG and DBCO can stably exist after a large amount of literature review, experimental exploration and years of experience accumulation. On the one hand, DBCO can be easily labeled on the second protein through the specific reaction of primary amines and NHS esters; on the other hand, DBCOTAG has only 7 amino acids and can be fused with the first protein without much effect on the original properties of the protein. After the reaction between DBCO and DBCOTAG, the first protein (eg, luciferase) and the second protein (eg, antibody) are coupled at a defined half-site.

[0094] "Half-site defined coupling" means that when a binding reaction occurs between two biomacromolecules (such as proteins), only the binding site of one of the biomacromolecules is fixed, while the binding site of the other biomacromolecule is not determined or fixed. In the present application, the half-site defined coupling of a first protein and a second protein means that only the binding site of the first protein is fixed, while the binding site of the second protein is not determined.

[0095] Prior art

[0096] The existing conjugation method involves the use of SMCC (4-(N-maleimido) cyclohexane-1-carboxylate succinimidyl) to react with the primary amine groups on Nano luciferase (Nluc), followed by the use of tris(2-carboxyethyl)phosphine hydrochloride (TECP) to reduce the digoxigenin antibody (Ab-Dig) to expose its free sulfhydryl groups. Subsequently, Nluc is cross-linked with Ab-Dig through the maleimido group on the SMCC molecule to form the Nluc-Ab-Dig conjugated product. It should be noted that Nluc has a β-barrel structure with only one non-conserved cysteine ​​and no disulfide bonds within the molecule. In contrast, Gluc contains 10 cysteines and 5 pairs of disulfide bonds are formed within the molecule. Due to these differences in the molecular structure of Nluc and Gluc, it is not possible to ensure that the Gluc conjugated product has similar enzymatic activity to Nluc. In addition, since only one molecular linker is used in the conjugation process, the experimental process is linear, which significantly reduces the flexibility of experimental design during development. It should be pointed out that the maleimide group in the SMCC molecule needs to react with the free sulfhydryl group in the protein, which will inevitably destroy the natural structure of the protein, resulting in a decrease in the natural activity of the protein or even inactivation. In addition, one of the crosslinkers needs to react with the primary amines on the surface of luciferase, and there are many primary amines on the surface of luciferase, which will inevitably destroy the original structure and properties of the protein and reduce the enzyme activity. Since the distribution of primary amines on the surface of proteins is relatively random, the labeling position often has a large uncertainty, so the structure of the product is often difficult to predict in advance.

[0097] Protein coupling method

[0098] Therefore, the present application develops a protein coupling method with a determined half-site to solve the problems of reduced enzyme activity of the coupling product and linearization of the experimental process.

[0099] Specifically, the method comprises: coupling a first protein with a second protein, wherein the first protein is connected to DBCOTAG, and the second protein is connected to DBCO. Compared with the existing thiol coupling method, the product has high stability and high activity.

[0100] In some examples of the present application, the aforementioned coupling method can also be applied to the field of gene sequencing, the field of molecular detection and western-blot experiments.

[0101] In the field of gene sequencing, the coupling method uses the signal detection of the E-series sequencer of the BGI Group. This method produces a new type of protein marker by labeling different bases with digoxin or biotin and using the corresponding antibody conjugates for recognition, which can be widely used in the field of sequencing. For example, the T base is labeled with digoxin, the A base is labeled with biotin, the C base is labeled with digoxin and biotin, and the G base is unlabeled. The T base and C base labeled with digoxin are recognized by the luciferase-digoxin antibody conjugate that couples to recognize the FCTZ substrate, while the A and C bases labeled with biotin are recognized by the luciferin-streptavidin conjugate that recognizes CTZ.

[0102] In the field of molecular detection, this method can be used to improve the double antibody sandwich ELISA. The antibody A of the antigen to be tested is marked on the ELISA plate, and then the coupling method is used to combine luciferase with the second antibody of antibody B to achieve high sensitivity detection of the antigen to be tested, rather than just the traditional HRP labeling method. For example, the antibody A of the antigen to be tested is marked on the ELISA plate, the antigen to be tested is added, and the antigen is captured by the antibody A on the ELISA plate, and then the antibody B of the antigen to be tested is added, and antibody B is captured by the antigen to be tested on the ELISA plate, and then the method described in this invention is used to couple luciferase with the second antibody of antibody B, and add the above-mentioned ELISA plate, and the conjugate will be captured by antibody B, and the amount of the antigen to be tested can be reflected by the light signal by adding luciferase substrate. The second antibody used in this scheme is generally HRP (catalase) labeled second antibody at present, and HRP catalyzes TMB substrate to generate OX-TMB, and OX-TMB color is darker and can be detected by absorption light of different wavelengths.

[0103] In Western-blot experiments, the coupling method can also be used to improve the experiment. The luciferase-antibody conjugate produced by the technical solution of the present application can also replace the ERP-labeled secondary antibody. The general detection method is to transfer the protein to be tested to a PVDF membrane, incubate the corresponding primary antibody and the ERP-labeled secondary antibody, and detect with ECL luminescent liquid. The principle of ECL luminescence is that ERP oxidizes the chemiluminescent substance luminol to emit light in the presence of hydrogen peroxide. After replacing with luciferase, the luciferase substrate can be used to replace the ECL luminescent liquid. The luciferase luminescence system usually emits brighter light than the ERP-luminol luminescence system, and the wavelength range is wider.

[0104] The present invention is described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used without indicating the manufacturer are all conventional products that can be obtained commercially.

[0105] Example 1: Co-expression and purification of luciferase and DBCOTAG

[0106] 1. Cell culture and plasmid transformation

[0107] 1.1 Thaw the Origami DE3 competent cells on ice and aliquot into 50 μl tubes;

[0108] 1.2 Add 5 μl of plasmid to each tube of cell suspension and mix gently;

[0109] 1.3 Place on ice for 1 hour, then heat shock at 42°C for 1 minute, and place on ice again for 5 minutes;

[0110] 1.4 Add 500 μl of LB medium without antibiotics and incubate at 37°C, 220 rpm for 1 hour;

[0111] 1.5 Take 60 μl of bacterial solution and spread it evenly on LB agar plate containing Ampicillin antibiotics, and culture it at 37°C overnight.

[0112] 2. Protein expression and purification

[0113] 2.1 Pick a single clone from the overnight culture plate and then grow it in LB broth containing Ampicillin antibiotics at 37°C, 220 rpm until OD600 reaches 0.6;

[0114] 2.2 The bacteria were expanded at a ratio of 50:1 and transferred to LB medium containing ampicillin in a suitable experimental volume, and shaken at 37°C, 200 rpm for 3 to 4 hours until the OD600 reached 0.6;

[0115] 2.3 Cool on ice for 15 minutes, then add 1 mM IPTG to a final concentration of 1 μM, and continue to induce expression overnight at 16°C, 300 rpm;

[0116] 2.4 Centrifuge the bacterial solution at 4000g for 10 minutes and remove the supernatant;

[0117] 2.5 Use pre-cooled PBS buffer containing 1mM PMSF to suspend the bacterial solution, and then use ultrasound to break the bacteria until clear;

[0118] 2.6 Centrifuge at 12000 g for 30 min at 4°C, retain the supernatant and store on ice for later use.

[0119] 3. Filler pretreatment and protein purification

[0120] 3.1 Rinse with sterile deionized water for 5CV, then with 1*PBS for 5CV, and finally with 1M imidazole for 5CV;

[0121] 3.2 Use 20-30 mM imidazole to flush 5CV to equilibrate the column;

[0122] 3.3 Add the treated filler to the treated sample and shake gently at 4°C for 30 minutes to 1 hour;

[0123] 3.4 Move the sample-filler mixture up to an empty column and retain the filler;

[0124] 3.5 Wash the filler with 1*PBS for 5CV, and then wash the filler with 20mM imidazole for 5CV;

[0125] 3.6 Use 300mM imidazole for elution, and the eluate is the target protein;

[0126] 3.7 Wash the packing material with 1 mM imidazole for 5 CV, and then wash the packing material with water for 5 CV;

[0127] 3.8 Finally, add 20% ethanol and store at 4°C.

[0128] 4. Ultrafiltration fluid replacement

[0129] 4.1 Transfer the purified protein to a 3k or 10k ultrafiltration tube (Merck );

[0130] 4.2 Add 10 times the volume of 1*PBS pre-cooled at 4℃;

[0131] 4.3 Centrifuge at 3900 g for 15 min at 4°C, repeat 3 times.

[0132] The results are as follows Figure 1 As shown, the co-expression and purification product of luciferase and DBCOTAG was obtained.

[0133] Example 2: Exploration of coupling conditions

[0134] To explore the optimal concentration of DTT, a concentration gradient of 0.01, 0.1, 1 and 10 mM DTT was used to treat Gluc-DBCOTAG for 1 h, and DTT was removed by ultrafiltration before gel running and enzyme activity detection. To explore whether DTT affects the activity of monomeric Gluc, a gradient of 0.01, 0.1 and 1 mM DTT was used to treat monomeric Gluc (MGI) in the same way, and the enzyme activity was detected. The results are shown in Figure 2 As shown, it shows that 0.1mM DTT treatment for 1h can fully disaggregate Gluc-DBCOTAG. 0.01-0.1mM DTT will not have a significant effect on Gluc activity, especially after the removal of DTT, while 1mM DTT has a greater effect on enzyme activity.

[0135] Take out the unopened DBCO-NHS (50 mg) from -20℃ and place it at room temperature to equilibrate the temperature for 10 minutes. Add 12.4254 ml of anhydrous DMSO (Sangong), vortex to mix, prepare a 10 mM storage solution and place it at -20℃ for use. Take Ab-Dig (MGI) with a concentration of 3.4 mg / ml and mix it evenly with different volumes of DBCO-HNS 10 mM storage solution (see Table 1 for the ratio), react at room temperature for 30 minutes, invert and mix every 10 minutes, and observe the precipitation state of the product after the reaction ( Figure 3 ). When the ratio of DBCO-HNS to Ab-Dig is less than 20, the protein does not precipitate.

[0136] Table 1

[0137]

[0138] N3-modified Gluc was mixed with DBCO-modified Ab-Dig at a molar ratio of 20:1 with DBCO:Ab-Dig (molar ratio) of 5, 10, 15, and 20, respectively, and reacted at 4°C overnight. Unreacted Gluc was removed with a 50k ultrafiltration tube and the enzyme activity was detected. Protein quantification was performed using a BCA kit, and it was diluted to 0.5pM with luciferase diluent (50mM Tris-HCl pH 8.0, 100mM NaCl, 0.1% (v / v) Tween-20), and 10ul of the diluted product was added to each well of a black 96-well plate. The Gluc substrate CTZ was diluted to 50uM with diluent and placed on ice for later use. The luminescence intensity was detected using the perfusion program of the microplate reader, 100μl of substrate was added to each well, and the luminescence was detected immediately. The luminescence gain was adjusted to 75, and the data was recorded and analyzed. Results ( Figure 4 ) showed that the enzyme activity was highest when the ratio of DBCO-HNS to Ab-Dig was 5.

[0139] Example 3: Conjugation of luciferase and antibodies

[0140] 1. Antibody DBCO treatment steps

[0141] 1.1 Take out the unopened DBCO-NHS (50 mg) from the refrigerator and place it at room temperature for about 10 minutes;

[0142] 1.2 Add 12.4254 ml of anhydrous DMSO (Sangon), vortex to mix, and prepare a 10 mM stock solution;

[0143] 1.3 Mix the prepared DBCO-NHS 10mM stock solution with Ab-Dig at a molar ratio of 1:5, react at room temperature for 30 minutes, and mix by inversion every 10 minutes;

[0144] 1.4 Transfer the reaction product to a 30k or 50k ultrafiltration tube (Merck ), add 10 times the volume of 4°C pre-cooled PBS, centrifuge at 3900 rpm at 4°C for 15 min, and repeat 3 times.

[0145] 2. Pretreatment before Gluc coupling

[0146] 2.1 Add DTT to the Gluc obtained in Example 1 to a final concentration of 0.1 mM, and perform reduction at 25°C for 1 hour;

[0147] 2.2 Transfer the reaction product to a 3k ultrafiltration tube (Merck ), add 10 times the volume of 4°C pre-cooled PBS, centrifuge at 3900 rpm at 4°C for 15 min, and repeat 3 times to remove DTT.

[0148] 3. Coupling reaction

[0149] 3.1 Mix the treated Gluc and antibody at a molar ratio of 10:1 and react at 37°C overnight. Figure 5 shown.

[0150] 4. Purification

[0151] 4.1 Use the Protein A column purification method to remove excess unreacted Gluc, and mix the Protein A filler with the coupling product sample at a ratio of sample / Protein A filler volume = 100:1;

[0152] 4.2 Place on an ice box and mix by turning it upside down every 5 minutes for a total of 30 minutes;

[0153] 4.3 Take an empty affinity chromatography column and manually pass the coupling product with Protein A filler through the column;

[0154] 4.4 Rinse the column with 1*PBS for 10CV, then elute the target protein with Protein A purification solution B (containing 100mM glycine, 20mM arginine, pH 3.0), and collect 6-8 tubes of eluate in total;

[0155] 4.5 After elution, wash the pre-packed column with 5CV 1*PBS, then rinse with Milli Q H2O for 5CV, and finally rinse with 20% ethanol for 5CV, and store the filler in 20% ethanol.

[0156] The results are as follows Figure 6 As shown, the resulting Gluc-AbDIG conjugated product was purified.

[0157] Example 4: Activity detection of coupling products

[0158] 1. Chemiluminescence method steps

[0159] 1.1 Quantify the purified protein and perform electrophoresis;

[0160] 1.2 After electrophoresis, soak the gel in deionized water for 10 minutes, then wash the gel with 25% isopropanol for 3 minutes, and repeat the washing twice;

[0161] 1.3 Then wash the gel with deionized water for 3 minutes, twice in total;

[0162] 1.4 Soak the gel in 5 mL of luciferase dilution solution (containing 50 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.1% (v / v) Tween-20) for 3 minutes;

[0163] 1.5 Add 100 μL of 5 mM CTZ to the system and rinse for 1 minute;

[0164] 1.6 Detect luminescence using a chemiluminescent gel imager.

[0165] After detection, the protein gel was stained with Coomassie Brilliant Blue to detect the protein. Figure 7 As shown, the coupled product was obtained.

[0166] 2. Protease activity detection steps

[0167] 2.1 Protein quantification using BCA kit;

[0168] 2.2 Use luciferase diluent (including 50mM Tris-HCl pH 8.0, 100mM NaCl, 0.1% (v / v) Tween-20) to dilute it to 0.5pM;

[0169] 2.3 In a black 96-well plate, add 10 μl of diluted product to each well;

[0170] 2.4 Dilute the Gluc substrate CTZ to 50 μM with diluent and place on ice for later use;

[0171] 2.5 Use the perfusion program of the microplate reader to detect the luminescence intensity. Add 100 μl of substrate to each well and detect the luminescence immediately. The luminescence gain is adjusted to 75.

[0172] 2.6 Record data and analyze them.

[0173] The results are as follows Figure 8 As shown, there was no significant difference between the activity of the coupled product (Gluc-AbDIG) and the luciferase (Gluc) activity.

[0174] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0175] The above embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present application. It should be pointed out that, for a person of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the present application shall be subject to the attached claims.

Claims

1. A protein coupling method, characterized in that: include: The first protein is coupled to the second protein, wherein the first protein is connected to DBCOTAG, and the second protein is connected to DBCO.

2. The method according to claim 1, characterized in that The second protein is connected to DBCO via an amide bond or coupled via a sulfhydryl group; Preferably, the second protein is linked to DBCO via an amide bond; Preferably, the second protein is linked to DBCO via an amide bond by the following method: The second protein is contacted with DBCO linked with N-hydroxysuccinimide, wherein the second protein has free -NH2.

3. The method according to claim 2, characterized in that In the contact system, the molar ratio of the second protein to DBCO connected with N-hydroxysuccinimide is 1:5 to 1:

20.

4. The method according to claim 1, characterized in that: Before the coupling treatment, the first protein is further reduced; Optionally, the reduction treatment is carried out in the presence of a reducing agent; Optionally, the reducing agent comprises at least one selected from DTT, β-mercaptoethanol and TCEP.

5. The method according to claim 1, characterized in that In the coupling treatment system, the molar ratio of the first protein to the second protein is 5:1 to 1:

1.

6. The method according to claim 2, characterized in that The DBCO linked to N-hydroxysuccinimide is provided in the form of DBCO-(PEG)n-NHS, wherein n is an integer between 0 and 12; Preferably, n is 0, 4, 8 or 12.

7. The method according to claim 1, characterized in that The N-terminus or C-terminus of the first protein is connected to DBCOTAG; Preferably, the C-terminus of the first protein is connected to the N-terminus of the DBCOTAG.

8. The method according to claim 7, characterized in that The N-terminus or C-terminus of the first protein is connected to DBCOTAG via a connecting peptide; Optionally, the connecting peptide comprises at least one selected from a flexible peptide and a rigid peptide; Optionally, the amino acid sequence of the flexible peptide is (GGGGS) n1 ; wherein n1 is an integer greater than or equal to 1, preferably 1, 2 or 3; Optionally, the amino acid sequence of the rigid peptide is (EAAAK) n2 ; wherein n2 is an integer greater than or equal to 1, preferably 1, 2 or 3.

9. The method according to claim 1, characterized in that: The N-terminus or C-terminus of the first protein is further connected with a His tag, preferably, the His tag is 6*His.

10. The method according to claim 9, characterized in that The N-terminus of the first protein is connected to a His tag, the C-terminus of the DBCOTAG is connected to the N-terminus of the His tag, or the N-terminus of the DBCOTAG is connected to the C-terminus of the first protein; Preferably, the N-terminus of the DBCOTAG is connected to the C-terminus of the first protein.

11. The method according to claim 9, characterized in that The C-terminus of the first protein is connected to a His tag, the C-terminus of the DBCOTAG is connected to the N-terminus of the first protein, or the N-terminus of the DBCOTAG is connected to the C-terminus of the His tag; Preferably, the N-terminus of the DBCOTAG is connected to the C-terminus of the His tag.

12. The method according to claim 9 or 10, characterized in that: The C-terminus of the DBCOTAG is connected to the N-terminus of the His tag, the C-terminus of the His tag is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the first protein; or The C-terminus of the His tag is connected to the N-terminus of the first protein, the C-terminus of the first protein is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the DBCOTAG.

13. The method according to claim 9 or 11, characterized in that: The C-terminus of the DBCOTAG is connected to the N-terminus of the first protein, the C-terminus of the first protein is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the His tag; or The C-terminus of the first protein is connected to the N-terminus of the connecting peptide, the C-terminus of the connecting peptide is connected to the N-terminus of the His tag, and the C-terminus of the His tag is connected to the N-terminus of the DBCOTAG.

14. The method according to claim 1, characterized in that The first protein and the second protein are independently selected from luciferase and antibodies, respectively.

15. The method according to claim 14, characterized in that The luciferase comprises at least one selected from Gaussia luciferase or mutant, Nano luciferase or mutant, bacterial luciferase or mutant, firefly luciferase or mutant, pyralis luciferase or mutant, Renilla luciferase or mutant, and spiny shrimp luciferase or mutant; Preferably, the luciferase is Gaussia luciferase.

16. The method according to claim 14, characterized in that The antibody is selected from at least one of digoxin antibody, A base antibody, G base antibody, C base antibody, U base antibody or T base antibody; Optionally, the antibody is selected from digoxin antibodies.

17. A protein complex, characterized in that The protein complex is prepared by the method according to any one of claims 1 to 16.

18. A protein complex, characterized in that include: A first protein, wherein the first protein is linked to DBCOTAG, preferably, the N-terminus or C-terminus of the first protein is linked to DBCOTAG; as well as A second protein, wherein the second protein is connected to DBCO, and the DBCOTAG is connected to the DBCO via a carbon-sulfur bond; Optionally, the second protein is linked to the DBCO via an amide bond; Optionally, the first protein and the second protein are independently selected from luciferase and antibodies, respectively.

19. A molecular detection method, characterized in that include: contacting the molecule to be tested with the protein complex according to claim 17 or 18, wherein the first protein and the second protein are independently selected from luciferase and antibody; determining the molecule based on the signal after the contacting treatment; Wherein, the molecule to be detected carries the luciferase substrate or has binding activity with the antibody.

20. A method for determining the position of a target molecule, characterized in that: include: contacting the target molecule with the protein complex according to claim 17 or 18, wherein the first protein and the second protein are independently selected from luciferase and an antibody; Determining the location of the target molecule based on the signal after the contacting process; Wherein, the target molecule carries a luciferase substrate or has binding activity with an antibody.

21. The method according to claim 19 or 20, characterized in that The signal is a fluorescent signal.

22. A kit, characterized in that: Comprising the protein complex of claim 18 or 19.

23. The kit according to claim 22, characterized in that Further comprising at least one of a luciferase substrate and a luciferase buffer.

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