KR102159737B1 - Streptactin conjugated quantum dot and process for preparing the same - Google Patents

Abstract

The present invention provides a quantum dot-polyethylene glycol linker-biotin-streptactin-conjugated quantum dot having a streptactin structure, a use of the protein labeling/imaging thereof, and a method for producing the same.
Streptactin-conjugated quantum dots of the present invention, the use of protein labeling/imaging thereof, and a method for preparing the same can be usefully used as protein labeling and molecular imaging techniques in the field of protein recombination and biotechnology.

Description

Streptactin conjugated quantum dot and process for preparing the same}

The present invention relates to a streptactin-conjugated quantum dot and a method for manufacturing the same, and more particularly, to a streptactin-conjugated quantum dot and a method for producing the same, in which protein affinity is improved by introducing a PEG linker.

Avidin is a protein derived from white, and streptavidin is a protein derived from Streptomyces avidinii. Avidin or streptavidin has a very high affinity with biotin (Kd = 10 ^{-15 to -14} M), and as an interaction between two molecules in a living body, it is one of the strongest interactions, and its molecular weight is about 60 kDa. Currently, the avidin/streptavidin-biotin interaction is widely applied in the fields of biochemistry, molecular biology, or medicine. This very strong and specific interaction between streptavidin or avidin and biotin has been applied in a variety of applications such as bio-interfaces and molecular labeling for nanostructure assembly.

On the other hand, a fluorescence microscope (FLM) uses the principle of emitting fluorescence when a phosphor absorbs light of a specific wavelength. After applying a fluorescent material to a sample sample, cooling it with liquid nitrogen, It is a device that observes a sample through radiation light emitted from the sample by irradiating light at the absorption wavelength of the material. Fluorescence microscopes can be effectively used for samples in which a sample itself has fluorescence or can adsorb a fluorescent material. Accordingly, it is widely used for testing biological materials such as bacteria and proteins or biological samples.

As described above, technologies for detecting a target substance in a sample or imaging a protein produced by recombinant technology are used in various fields, and fluorescent nanoparticles are used for intracellular protein analysis using imaging equipment. Conventionally, quantum dots [Qdot® ITK™ streptavidin quantum dots, (A10196, Thermo)] using streptavidin-biotin bonds have been used as a means of detection/imaging of recombinant proteins using these fluorescent nanoparticles, but targets due to structural limitations There is a problem in that molecular imaging efficiency is low due to low affinity for proteins.

Korean Patent Publication No. 10-2018-0108567 (2018.10.04.) US published patent US2011165647A1 (2011.07.07.) US published patent US20170212121A1 (2017.07.27.)

The inventors of the present invention have conventionally used fluorescent nanoparticles for intracellular protein analysis and cell imaging techniques, but the affinity for the protein to be imaged is low, so while studying the quantum dot structure to improve this, biotin-modified quantum dots It was found that the functionalized quantum dots in which streptactin was bound by using PEG as a linker was significantly improved compared to the existing streptavidin-bound quantum dots.

Accordingly, an object of the present invention is to provide a quantum dot-polyethylene glycol linker-biotin-streptactin-conjugated quantum dot having a streptactin structure, a use of molecular imaging thereof, and a method for producing the same.

According to an aspect of the present invention, a quantum dot-polyethylene glycol linker-biotin-streptactin-conjugated quantum dot having a streptactin structure is provided.

In one embodiment, the polyethylene glycol linker may have -CH ₂ CH ₂ O- in 5 to 15 integer units.

In one embodiment, the streptactin may be a tetramer.

According to another aspect of the present invention, a composition for labeling a protein comprising the quantum dots conjugated to streptactin and a method for labeling proteins using the quantum dots conjugated to streptactin are provided.

According to another aspect of the present invention, (a) modifying biotin including a polyethylene glycol linker in the quantum dot; And (b) there is provided a method of manufacturing streptactin-conjugated quantum dots comprising the step of conjugating streptactin to the biotin-modified quantum dots.

In one embodiment, step (a) may be performed in a borate buffer.

In one embodiment, the reaction ratio of biotin: quantum dots in step (a) may be 20 to 30.

In one embodiment, the biotin-modified quantum dots in step (b) may have a concentration of 50 to 500 pM.

In one embodiment, the streptactin of step (b) may be used in a molar concentration ratio of 100 to 20000 times that of biotin-modified quantum dots.

In one embodiment, in step (b), BSA and Tween 20 may be additionally added and performed.

According to the present invention, the streptactin-conjugated quantum dot having a quantum dot-polyethylene glycol linker-biotin-streptactin structure is a quantum dot (Qdot) capable of simultaneous fluorescent labeling of a fluorescence microscope and structural indicators of an electron microscope, and antibody-PEG (Ab-PEG). ), and it shows a 1000-fold increase in affinity compared to the existing streptavidin-binding quantum dots, so that the labeling of the target protein in molecular imaging is performed by linked microscopy technique and Immuno-electron microscopy. It turns out that it can be used as a technique for

Therefore, the quantum dot-polyethylene glycol linker-biotin-streptactin-conjugated quantum dot having the structure of the present invention, its use for protein labeling, and its preparation method are useful as protein labeling and molecular imaging technology in the field of protein recombination and biotechnology. Can be used.

1 is a schematic diagram of a process of formulating biotin using EDC [1-ethyl-3-(3-dimethylaminopropyl) carbodiimide], which is a zero-length crosslinker in a carboxyl group on the surface of a quantum dot.
Figure 2 schematically shows the structure of a streptactin-functionalized quantum dot conjugate of the present invention.
3 shows the results of a gel shift assay according to the buffer composition and reactant ratio when biotin is modified in quantum dots. When PBS is used as a buffer (a), a borate buffer and MES (2-( In the case of using N-morpholino)ethanesulfonic acid) buffer (b), in the step of formulating using 3,000 times more EDC for 250 nM carboxyl quantum dots 625, while controlling the reaction ratio of biotin to the quantum dots (25 ~ 500 times) This is the result of confirming the change in the amount of biotin actually bound to the surface through the HABA assay (HABA/avidin reagent, Sigma) (c).
4 is a result of confirming the result of conjugation of streptactin to biotin-modified quantum dots using a gel shift assay to confirm that the conjugation of streptactin protein was achieved. The conjugation reaction was carried out in 50 mM borate, pH 8.3 buffer, and the concentration of the quantum dots was 100 pM during the reaction, and the streptactin was reacted at a molar ratio of 500 times compared to the bound biotin. This was carried out by adding mg/ml bovine serum albumin (BSA) and 0.05% Tween-20.
5 is a fluorescence image of cells obtained using (a) conventional streptavidin-biotin-binding quantum dots (commercial material, A10196, Thermo), (b) functionalized quantum dots of the present invention [polyethylene glycol linker-biotin-streptactin Fluorescence images of cells obtained using [Coupled Quantum Dots], (c) Immuno-electron microscopy images obtained using the functionalized quantum dots of the present invention.

In the present specification, the term "streptactin" refers to an oligomeric mutant streptavidin as an analog of streptavidin, and is commercially available as strep-tactin ^® (Strep-Tactin ^® ). It is a protein that has strong binding power to biotin.

According to an aspect of the present invention, a quantum dot-polyethylene glycol linker-biotin-streptactin-conjugated quantum dot having a streptactin structure is provided.

Quantum dots (QD) used in the present invention may use commonly used quantum dots, preferably QD 625. The streptactin-conjugated quantum dot of the present invention is a functionalized quantum dot composed of a structure of quantum dot-PEG-biotin-streptactin, and the polyethylene glycol linker contains -CH ₂ CH ₂ O- in 5 to 15 integer units, preferably 9 to 13 It may have an integer unit, most preferably 11 integer units. In addition, the streptactin may be a tetramer.

According to another aspect of the present invention, a composition for labeling a protein comprising the quantum dots conjugated to streptactin and a method for labeling proteins using the quantum dots conjugated to streptactin are provided. The functionalized quantum dots made of the structure of the QD-PEG-biotin-streptactin of the present invention exhibit an affinity increase of about 1000 times compared to the existing streptavidin-binding quantum dots, and thus can be usefully used in the field of protein recombination and detection/imaging technology. .

According to another aspect of the present invention, (a) modifying biotin including a polyethylene glycol linker in the quantum dot; And (b) there is provided a method of manufacturing streptactin-conjugated quantum dots comprising the step of conjugating streptactin to the biotin-modified quantum dots.

In the manufacturing method of the present invention, a chemical binding reaction between carboxyl QD and EDC and amine-PEG-biotin is performed, and a process of attaching a streptactin protein to the biotin-modified QD is performed to prepare streptactin-conjugated quantum dots. I can. QD is preferably QD 625, and can be performed by determining the optimum buffer composition and reactant ratio in each reaction step, and whether or not streptactin protein is conjugated to the prepared final quantum dot is determined by using a gel shift assay. Can be used to check.

In one embodiment, the step (a) may be performed in a borate buffer, preferably 1 to 50 mM borate, pH 7.0 to 7.8, and more preferably 10 mM borate ( borate), pH 7.4 buffer.

In one embodiment, the reaction ratio of biotin: quantum dots in step (a) may be 20 to 30.

In one embodiment, the biotin-modified quantum dots in step (b) may have a concentration of 50 to 500 pM.

In one embodiment, the streptactin of step (b) may be used in a molar concentration ratio of 100 to 20000 times that of biotin-modified quantum dots. In this case, it is possible to prevent the agglomeration of quantum dots generated due to the tetramer structure of streptactin during the reaction process.

In one embodiment, in step (b), BSA and Tween 20 are additionally added as stabilizers to carry out the reaction.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are for illustrating the present invention, and the scope of the present invention is not limited thereto.

<Example>

1. Preparation of streptactin-conjugated quantum dots

(1) Formulation of biotin on the surface of carboxyl quantum dots (QDot)

The chemical bonding reaction between the carboxyl group on the surface of the Qdot ITK carboxyl quantum dot 625 (Invitrogen) and the amino group of biotin (Amine-PEG ₁₁ -biotin, Thermo) having a PEG linker was carried out as a crosslinker, EDC It was carried out using (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, Thermo) (see Fig. 1). In this process, each material stably exists and an optimal buffer in which the reaction occurs well was found, and finally, a buffer of 10 mM borate and pH 7.4 was selected.

In the above buffer conditions, biotin formulated using 3,000 times as many EDCs in 250 nM carboxyl quantum dots 625: while controlling the reaction ratio (25 to 500 times) of the quantum dots, the change in the number of biotin actually bound to the quantum dot surface was determined by HABA assay (HABA). /avidin reagent, Sigma). The reaction time was carried out for 2 hours at room temperature, and then washed 5 times with a filter [Nanosep-100 K filter (Pall)] using a centrifuge to remove biotin that did not participate in the reaction. The solution used for washing was a 50 mM borate (pH 8.3) buffer, and when washing with this buffer, it was confirmed that quantum dots did not adhere to the filter surface and were stably washed. The biotin-quantum dot after the reaction was completed was stored at 4 °C by blocking light.

(2) Biotin-modified quantum dots and streptactin protein conjugation

Next, the step of immobilizing the streptactin protein on the biotin-modified quantum dots was performed (see FIG. 2). Streptactin is an analog of streptavidin, a protein that has a strong binding power to biotin. During the reaction process, an aggregation phenomenon occurred between quantum dots due to the tetramer structure of streptactin, and the reaction conditions were optimized to prevent this. The reaction was carried out in 50 mM borate, pH 8.3 buffer, the concentration of the quantum dots during the reaction was carried out at 100 pM, and streptactin was reacted at a molar ratio of 500 times that of the bound biotin. For stable reaction, 2 mg/ml BSA and 0.05% Tween-20 were added to the solution. The reaction was carried out for 1 hour at room temperature while gradually dropping the quantum dot solution using a stirrer, and then stored overnight at 4° C. without a stirrer. Thereafter, in order to remove streptactin that did not participate in the reaction, it was washed four times with a filter [Nanosep-100 K filter (Pall)] using a centrifuge. The washing buffer used was 50 mM borate, pH 8.3 with 2 mg/ml BSA and 0.05% Tween-20 added. This is the buffer that contains. The washed solution was filtered using a 0.22 μm centrifugal filter (Millipore), and then the light was blocked and stored at 4°C.

(3) gel shift assay

Whether each reaction proceeded well was confirmed through a gel shift assay. Using 0.5% agarose (Lonza), 0.5 X TBE gel, it was developed at 100 V for 20 minutes and then the fluorescence of the quantum dots was checked with a gel to confirm the reaction.

As shown in (a) and (b) of FIG. 3, it was confirmed that biotin formulation did not occur in PBS and MES (2-(N-morpholino)ethanesulfonic acid) buffer among the three buffers, and proceeded only in the borate buffer. This seems to have been influenced by the stability of the quantum dots in the buffer and the pH with high reaction efficiency.

(4) Biotin: Adjusts the amount of biotin bound according to the quantum dot ratio

As the biotin: quantum dot ratio increases to 25 to 500 times, the number of bound biotin increases accordingly, and the result that the gel shift degree is proportional is shown in FIG. 3(c). The HABA assay revealed that about 50% of the reaction rate actually binds to the quantum dots. In the subsequent reaction, biotin was fixed at a reaction rate of 25 times.

(5) Fabrication of streptactin-conjugated quantum dots

When proceeding with the optimized reaction conditions, it was confirmed through gel shift that streptactin-quantum dots were well made without aggregation (see FIG. 4). A relatively large gel shift can be confirmed due to the size of the streptactin protein. In addition, it can be seen that it is made well uniformly with a circle band.

2. Fluorescent labeling and immunoelectron microscopy imaging results

Fluorescent images and immunoelectron microscopy (Immuno-electron microscopy) imaging of cells were performed using the prepared streptactin-quantum dot, and the results are shown in FIG. 5. Fluorescent images of cells were obtained using conventional streptavidin-biotin-coupled quantum dots (commercial material, A10196, Thermo) as a comparative group. The fluorescence image of FIG. 5 is the result of washing 5 times after binding between the target material and streptactin-quantum dot.

As shown in Figure 5, the fluorescence image (a) of cells obtained using the currently commercially available streptavidin-biotin-binding quantum dots (commercial material, A10196, Thermo) is a non-specific interaction within and outside the cell. ), it can be seen that it is difficult to clearly distinguish the target protein from the fluorescence image. On the other hand, in the fluorescence image (b) using the streptactin-quantum dot of the present invention, the target imaging point (indicated by the white arrow) of the target protein can be clearly identified, and the immunoelectron microscopy (Immuno-electron microscopy) obtained at a higher magnification , Immuno-EM) In the result (c), it was confirmed that the target protein was present through the presence of quantum dots around the mitochondria (black dots indicated by white arrows) and electron density around the quantum dots.

Claims (11)

delete delete delete Quantum dot-polyethylene glycol linker-biotin-streptactin-conjugated quantum dots having a streptactin structure for labeling proteins. Quantum dot-polyethylene glycol linker-biotin-streptactin-conjugated quantum dots having a streptactin structure. (a) modifying biotin containing a polyethylene glycol linker in the quantum dots; And
(b) conjugating streptactin to biotin-modified quantum dots
The method of manufacturing the composition of claim 4 comprising a. The method of claim 6, wherein step (a) is carried out in a borate buffer. The method of claim 6, wherein the reaction ratio of biotin: quantum dots in step (a) is 20 to 30:1. The method of claim 6, wherein the biotin-modified quantum dots in step (b) have a concentration of 50 to 500 pM. The method of claim 6, wherein the streptactin in step (b) is used in a molar concentration ratio of 100 to 20000 times that of biotin-modified quantum dots. The method of claim 6, wherein in step (b), BSA and Tween 20 are additionally added and performed.

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