CN111978404A - Protein one-step directional immobilization method based on Halo-tag specific dehalogenation reaction - Google Patents
Protein one-step directional immobilization method based on Halo-tag specific dehalogenation reaction Download PDFInfo
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Abstract
The invention discloses a protein one-step directional immobilization method based on Halo-tag specificity dehalogenation reaction, which recombines Halo-tag gene to the inactive C terminal of target receptor protein, modifies halogenated alkanoic acid to the surface of separation medium through acylation reaction, induces and expresses, and utilizes the specificity dehalogenation reaction of dehalogenase at the C terminal of recombinant receptor and halogenated alkane on the surface of separation medium to realize the one-step directional covalent immobilization of Halo-tag recombinant receptor in cell lysate, establishes a new protein one-step directional immobilization method, and can realize the one-step high specificity immobilization of protein through cell lysate, thereby effectively solving the problems of long protein immobilization period and low specificity of protein immobilization method.
Description
Technical Field
The invention relates to a protein immobilization method, in particular to a one-step directional protein immobilization method based on halogenated alkane dehalogenase (Halo-tag) specific dehalogenation reaction, and belongs to the technical field of protein immobilization.
Background
At present, the protein immobilization methods reported in the literature can be divided into: physical adsorption, chemical bonding and immobilization based on specific label affinity. In the above three methods:
(1) the physical adsorption method is to fix proteins on the surface of a solid matrix by non-specific adsorption. Although this method is most convenient in the process of immobilizing a protein, since this method is a method of immobilizing a protein on the surface of a solid material by nonspecific adsorption, the selectivity for the target protein is low, and the target protein must be first purified in the immobilization, the method often has disadvantages of long operation cycle and complicated operation.
(2) The chemical bonding method is to fix the protein on the surface of the carrier by chemical reaction with amino, carboxyl, sulfhydryl and the like exposed on the surface of the protein. The method also needs a large amount of high-purity protein, and the active site of the immobilized protein is easy to mask, so that the loss of the activity of the protein is large.
(3) The immobilization method based on the affinity of the specific tag is to recombine the common protein tags (such as a histidine tag, a glutathione transferase tag, a maltose binding protein tag, a streptavidin tag and the like) at the inactive tail end of the target protein, and realize the immobilization of the target protein by utilizing the specific affinity between the protein tags and the ligands thereof. The method needs to separate and purify the target protein, and the single label is difficult to realize the fine separation of the target protein, and often needs to be matched with ion exchange chromatography or size exclusion chromatography for further separation.
It is known that the protein immobilization process should have the following characteristics:
1. the specificity is strong, namely the nonspecific adsorption effect is weakened as much as possible, and the interference of impurity protein is eliminated;
2. the purification process of the protein is simplified as much as possible, and the loss of the protein is reduced;
3. the method of immobilizing the protein should be reliable, controllable and repeatable;
4. the active sites of the protein are exposed as much as possible to increase the number of bound drug molecules.
Therefore, it is important to develop a novel protein immobilization method.
Haloalkane dehalogenase (Halo-tag) was evolved from the microbial Rhodococcus rhodochrous dehalogenase, which in a catalytic dehalogenation reaction, enzymatically active site Asp106 nucleophilically substitutes the halogen group in the haloalkane to form Asp ester, forming a covalent alkanase intermediate. His272 in the enzyme can catalyze the hydrolysis of the intermediate to regenerate dehalogenase and generate fatty alcohol. If His272 is mutated to Phe, etc., the covalent alkane-enzyme intermediate is not hydrolyzed. At present, a great deal of research shows that Halo-tag can directionally capture target protein in the process of separating and purifying protein, and therefore, Halo-tag can be used as an ideal protein tag to be fused and expressed to the inactive C-terminal or N-terminal of the target protein.
G protein-coupled receptors (GPCRs) are a general name of a large class of transmembrane proteins, and in the field of marketed international market drug sales, 20% of the first 50 drugs with the best sales are GPCRs which serve as drug action targets and are combined with the GPCRs to further start a cascade signal conduction path to exert drug effects.
Disclosure of Invention
The invention aims to provide a protein one-step directional immobilization method based on Halo-tag specific dehalogenation reaction, which can effectively solve the problems of low specificity and long period of the existing protein immobilization method.
In order to achieve the above object, the present invention adopts the following technical solutions:
a protein one-step directional immobilization method based on Halo-tag specific dehalogenation reaction is characterized by comprising the following steps:
step 1: separation medium modified by halogenated alkanoic acid
Modifying halogenated alkanoic acid on the surface of a separation medium to obtain a halogenated alkanoic acid modified separation medium;
step 2: one-step directional immobilization of Halo-tag fusion protein
The haloalkanoic acid-modified separation medium obtained from Step1 was incubated with Halo-tag fusion protein expressed in cell lysates for 15min in 5mM ammonium acetate solution at pH 7.0.
The method for one-Step directional immobilization of proteins based on Halo-tag specific dehalogenation reaction is characterized in that, in Step1, the halogenated alkanoic acid is: 6-chlorohexanoic acid, 6-bromohexanoic acid, 6-fluorohexanoic acid, 6-iodohexanoic acid or all the saturated acids of the abovementioned halogens; the separation medium is: hydroxylated, aminated, sulfhydrylated or carboxylated modified resin microspheres or porous microspheres.
The method for one-Step directional immobilization of protein based on Halo-tag specific dehalogenation reaction is characterized in that in Step1, the halogenated alkanoic acid is 6-chlorohexanoic acid, the separation medium is aminated porous microspheres, and the method for modifying 6-chlorohexanoic acid to the surfaces of the aminated porous microspheres specifically comprises the following steps:
6-Chlorohexanoic acid, aminated porous microspheres, and diisopropylethylamine were dissolved in DMF, followed by addition of HATU and stirring at room temperature for 2 h.
The method for one-Step directional immobilization of proteins based on Halo-tag specific dehalogenation reaction is characterized in that in Step2, the proteins are GPCRs, and the GPCRs comprise: rhodopsin-like receptors, the secretin receptor family, metabotropic glutamate receptors, fungal mating pheromone receptors, cyclic adenosine receptors, and all members of the Frizzled/Smoothened family, wherein the rhodopsin-like receptors in turn comprise: beta is a2-adrenergic receptors and angiotensin II receptor type I receptors.
The invention has the advantages that:
the invention provides a protein one-step directional immobilization method based on Halo-tag specificity dehalogenation reaction, which recombines Halo-tag gene to the inactive C terminal of target receptor protein (for example GPCRs), modifies halogenated alkanoic acid to the surface of separation medium through acylation reaction, induces and expresses, and utilizes the specificity dehalogenation reaction of dehalogenase at the C terminal of recombinant receptor and halogenated alkane on the surface of separation medium, thus realizing one-step directional covalent immobilization of Halo-tag recombinant receptor in cell lysate, establishing a new protein one-step directional immobilization method, and effectively solving the problems of long protein immobilization period and low specificity of the protein immobilization method because one-step high specificity immobilization of protein can be realized through cell lysate.
Drawings
FIG. 1 is a one-step immobilization reaction scheme for Halo-tag fusion GPCRs;
FIG. 2 is an immobilized his-tag fusion of beta2-an immuno scanning electron micrograph of AR microspheres;
FIG. 3 is an immobilized Halo-tag fusion beta2-an immuno scanning electron micrograph of AR microspheres;
FIG. 4 shows the immobilization of valsartan on AT1A characterization behavior map on the R chromatographic model;
FIG. 5 is the immobilization of candesartan on AT1Characterization behavior pattern on the R chromatographic model.
Detailed Description
The invention is described in detail below with reference to the figures and the embodiments.
Example 1
Referring to fig. 1, the method for one-step directional immobilization of protein based on Halo-tag specific dehalogenation provided by the invention specifically comprises the following steps:
step 1: separation medium modified by halogenated alkanoic acid
The halogenated alkanoic acid may be selected from: 6-chlorohexanoic acid, 6-bromohexanoic acid, 6-fluorohexanoic acid, 6-iodohexanoic acid, and all saturated acids of the above halogens.
The separation medium can be selected from: resin microspheres, porous microspheres, etc. modified by functional groups such as hydroxylation, amination, sulfhydrylation, carboxylation, etc.
In this example, 6-chlorohexanoic acid was selected as the haloalkanoic acid, and aminated porous microspheres were selected as the separation medium.
The process of modifying the aminated porous microsphere by 6-chlorohexanoic acid is as follows:
dissolving 6-chlorohexanoic acid (1.2eq), aminated porous microspheres (1.0eq) and diisopropylethylamine (3.0eq) in DMF (5mL), adding 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate (HATU, 1.2eq), and stirring at room temperature for 2h to modify 6-chlorohexanoic acid to the surface of the aminated porous microspheres to obtain the microspheres modified by 6-chlorohexanoic acid.
Step 2: one-step directional immobilization of Halo-tag fusion protein
The protein can be selected from GPCRs, including: rhodopsin-like receptors, the secretin receptor family, metabotropic glutamate receptors, fungal mating pheromone receptors, cyclic adenosine receptors, and all members of the Frizzled/Smoothened family, wherein the rhodopsin-like receptors in turn comprise: beta is a2-adrenoceptor (. beta.)2-AR) and angiotensin II receptor type I receptor (AT)1R)。
In this example, the protein is selected from the group consisting of β in GPCRs2-adrenoceptor (. beta.)2-AR)。
Halo-tag fusion beta in cell lysates2The one-step directional immobilization process of AR is as follows:
fusing beta with 6-chlorohexanoic acid modified microspheres (1.0eq) obtained from Step1 and Halo-tag expressed in cell lysate2-AR (1.0eq) was incubated for 15min in 5mM ammonium acetate solution at pH 7.0 to effect dehalogenation to β2One-step directional immobilization of the AR to give immobilized beta2-AR microspheres.
Example 2
Referring to fig. 1, the method for one-step directional immobilization of protein based on Halo-tag specific dehalogenation provided by the invention specifically comprises the following steps:
step 1: separation medium modified by halogenated alkanoic acid
In this example, 6-chlorohexanoic acid was selected as the haloalkanoic acid, and aminated porous microspheres were selected as the separation medium.
The process of modifying the aminated porous microsphere by 6-chlorohexanoic acid is as follows:
dissolving 6-chlorohexanoic acid (1.2eq), aminated porous microspheres (1.0eq) and diisopropylethylamine (3.0eq) in DMF (5mL), adding HATU (1.2eq), and stirring at room temperature for 2 hours to modify 6-chlorohexanoic acid on the surfaces of aminated porous microspheres to obtain microspheres modified by 6-chlorohexanoic acid.
Step 2: one-step directional immobilization of Halo-tag fusion protein
In this example, the protein is selected from angiotensin II receptor type I receptors (AT) in GPCRs1R)。
Halo-tag fusion of AT in cell lysate1The one-step directional immobilization process of R is as follows:
fusing AT with 6-chlorohexanoic acid modified microspheres (1.0eq) obtained from Step1 and Halo-tag expressed in cell lysate1Incubating R (1.0eq) in 5mM ammonium acetate solution AT pH 7.0 for 15min to effect dehalogenation reaction and AT1One-step directional immobilization of R to obtain immobilized AT1R microspheres.
Immobilization of beta from example 1 Using the ImmunoScan Electron microscopy technique2-AR microspheres for detection. The detection method specifically comprises the following steps:
the immobilized beta obtained in example 1 was taken2Putting 50mg of AR microspheres into a centrifuge tube, wherein the system of the replacement solution is 1 xTBST buffer solution, adding 500 mu L of sealing solution containing 5% skimmed milk powder into the centrifuge tube, incubating at room temperature for 1h, washing with 100 mu L of 1 xTBST buffer solution for 3 times, centrifuging to remove supernatant, and obtaining the immobilized beta after sealing2-AR microspheres, according to 1: dilution ratio of 200 with 1 XTSST buffer solution to obtain beta2-AR antibody solution, immobilizing the immobilized beta at 4 deg.C2-AR microspheres with 300. mu.L of beta2Incubating the-AR antibody solution overnight, washing with 1 XTSST buffer solution for 3 times, centrifuging to remove the precipitate, and obtaining the immobilized Halo-tag fusion beta after antibody incubation2-AR microspheres;
the immobilized his-tag fusion beta after antibody incubation is obtained by the same method2-AR microspheres;
1 × TBST buffer solution was used as per 1: 25, preparing an IgG colloidal gold antibody solution, and respectively fusing beta with immobilized Halo-tag incubated with the antibody2Immobilized his-tag fusion beta after incubation of-AR microspheres and antibody2Adding 250 mu L of IgG colloidal gold solution into the AR microspheres, incubating for 1h at room temperature, washing for 3 times by using 1 xTBST buffer solution, each time washing for 100 mu L, centrifuging to remove supernatant, precipitating, and drying overnight at 60 ℃ to respectively obtain corresponding microspheres;
and (3) determining the corresponding microspheres by adopting an immune scanning electron microscope technology.
The results of the immunoscan-sem measurements are shown in fig. 2 and 3.
As can be seen from fig. 2 and 3: microspheres in FIG. 2 (immobilized his-tag fusion. beta2-AR microspheres) smooth surface, whereas the microspheres in figure 3 (immobilized Halo-tag fusion β)2AR microspheres) are attached with white dots of IgG colloidal gold antibodies.
This proves that: the immobilization method provided by the invention has strong specificity and no non-specific adsorption effect, and the receptor still has specific antibody recognition activity after immobilization.
AT with AT1R-specific ligands Valsartan and Candesartan on the immobilized AT obtained in example 21And (5) performing characterization by using an R chromatographic model.
The characterization results are shown in fig. 4 and 5.
As can be seen from fig. 4 and 5: AT1Immobilization of two drugs of R-specific ligand valsartan and candesartan on AT1The chromatographic retention times on the chromatographic columns of the R microspheres are different and very different.
This indicates that: after being immobilized, the receptor still has the function of identifying specific ligands.
Furthermore, the immobilization method (Halo-tag method for short) provided by the present invention was compared with conventional immobilization methods (e.g., random immobilization method, chemical bonding method, and specific labeling method) by means of examples 1 and 2, and the comparison results are shown in Table 1.
TABLE 1 comparison of protein immobilization methods
As can be seen from table 1: the Halo-tag-based protein one-step directional immobilization method provided by the invention can realize rapid directional immobilization of the target protein without purifying the target protein, and the operation period of the protein immobilization process is obviously shortened.
In conclusion, the method provided by the invention has the advantages of short protein immobilization period, strong specificity, capability of realizing one-step rapid immobilization of the target protein without purifying the target protein, and the like.
It should be noted that the above-mentioned embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the protection scope of the present invention.
Claims (7)
1. A protein one-step directional immobilization method based on Halo-tag specific dehalogenation reaction is characterized by comprising the following steps:
step 1: separation medium modified by halogenated alkanoic acid
Modifying halogenated alkanoic acid on the surface of a separation medium to obtain a halogenated alkanoic acid modified separation medium;
step 2: one-step directional immobilization of Halo-tag fusion protein
The haloalkanoic acid-modified separation medium obtained from Step1 was incubated with Halo-tag fusion protein expressed in cell lysates for 15min in 5mM ammonium acetate solution at pH 7.0.
2. The method for one-Step directional immobilization of proteins based on Halo-tag specific dehalogenation reaction of claim 1 wherein, in Step1, the haloalkanoic acid is: 6-chlorohexanoic acid, 6-bromohexanoic acid, 6-fluorohexanoic acid, 6-iodohexanoic acid or all the saturated acids of the abovementioned halogens.
3. The method for the one-Step directional immobilization of proteins based on Halo-tag specific dehalogenation reaction of claim 1, wherein in Step1, the separation medium is: hydroxylated, aminated, sulfhydrylated or carboxylated modified resin microspheres or porous microspheres.
4. The method for one-Step directional immobilization of proteins based on Halo-tag specific dehalogenation reaction of claim 1, wherein in Step1, the haloalkanoic acid is 6-chlorohexanoic acid, the separation medium is aminated porous microspheres, and the method for modifying 6-chlorohexanoic acid to the surfaces of the aminated porous microspheres is as follows:
6-Chlorohexanoic acid, aminated porous microspheres, and diisopropylethylamine were dissolved in DMF, followed by addition of HATU and stirring at room temperature for 2 h.
5. The method for the one-Step directional immobilization of proteins based on Halo-tag specific dehalogenation reaction of claim 1 wherein in Step2 the proteins are GPCRs.
6. The method for one-step directional immobilization of proteins based on Halo-tag specific dehalogenation reaction of claim 5, wherein the GPCRs comprise: rhodopsin-like receptors, the secretin receptor family, metabotropic glutamate receptors, fungal mating pheromone receptors, cyclic adenosine receptors, and all members of the Frizzled/Smoothened family.
7. The method for one-step directional immobilization of proteins based on Halo-tag specific dehalogenation reaction of claim 6, wherein the rhodopsin-like receptor comprises: beta is a2-adrenergic receptors and angiotensin II receptor type I receptors.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113527504A (en) * | 2021-08-10 | 2021-10-22 | 苏州海澳斯凯尔生物科技有限公司 | Directional antibody immobilization method based on bioorthogonal reaction |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104844710A (en) * | 2015-04-13 | 2015-08-19 | 西北大学 | Preparation method and applications of oriented immobilized PEGA composite resin |
| CN107356748A (en) * | 2017-07-17 | 2017-11-17 | 广东志道医药科技有限公司 | Biphenthrin colloidal gold immuno-chromatography test paper strip and its preparation and application |
| CN108697732A (en) * | 2015-12-04 | 2018-10-23 | Npo佩特洛瓦克斯制药有限责任公司 | The method of hyaluronidase conjugate and the application of gained conjugate are prepared with polyethylene bridged piperazine derivatives |
-
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- 2020-08-11 CN CN202010802740.2A patent/CN111978404A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104844710A (en) * | 2015-04-13 | 2015-08-19 | 西北大学 | Preparation method and applications of oriented immobilized PEGA composite resin |
| CN108697732A (en) * | 2015-12-04 | 2018-10-23 | Npo佩特洛瓦克斯制药有限责任公司 | The method of hyaluronidase conjugate and the application of gained conjugate are prepared with polyethylene bridged piperazine derivatives |
| CN107356748A (en) * | 2017-07-17 | 2017-11-17 | 广东志道医药科技有限公司 | Biphenthrin colloidal gold immuno-chromatography test paper strip and its preparation and application |
Non-Patent Citations (1)
| Title |
|---|
| KAIZHU ZENG等: "One-step methodology for the direct covalent capture of GPCRs from complex matrices onto solid surfaces based on the bioorthogonal reaction between haloalkane dehalogenase and chloroalkanes", 《CHEMICAL SCIENCE》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113527504A (en) * | 2021-08-10 | 2021-10-22 | 苏州海澳斯凯尔生物科技有限公司 | Directional antibody immobilization method based on bioorthogonal reaction |
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