WO2024117805A1 - Amino acid tag for immobilization on carbon nanomaterials and use thereof - Google Patents

Abstract

The present invention relates to an amino acid tag for immobilization on carbon nanomaterials and a use thereof. Specifically, the tag for immobilization on carbon nanomaterials of the present invention can be effectively fixed to carbon nanomaterials such as graphene without the need for separate linkers or chemical treatments. Accordingly, the tag immobilizes a target protein fused to the C-terminus thereof on carbon nanomaterials, with orientation imparted into the protein, and thus can be advantageously used for constructing a biosensor that converts biological signals into electrochemical signals.

Description

Amino acid tags for immobilization on carbon nanomaterials and uses thereof

The present invention relates to amino acid tags for immobilization on carbon nanomaterials and their use.

A biosensor refers to an analysis device that detects an analyte generated from a biological element or the interaction between an analyte and a biological element through a physical or chemical signal transducer. A biosensor consists of a bio-receptor part for detecting analytes, a sensor part that detects the interaction between analytes and bio-receptors as physical and chemical signals, and a transducer that converts and amplifies the obtained signals into electrical signals. It can be broadly divided into (transducer) parts. At this time, bioreceptors include tissues, cells, enzymes, antibodies, nucleic acids, etc., and may be used by imitating conventional biological elements or improving their structure and function using biotechnology.

Detection methods used in biosensors include various physical and chemical technologies, including optical, electrochemical, and piezoelectric. Recently, various types of biosensors with portability, economic efficiency, wide detection limits, and multiple detection functions have been developed depending on the purpose. Meanwhile, conductive nanostructures such as conductive polymer nanoparticles, tubes, carbon nanorads, graphene, and fluorescent structures are used as converters, and research on graphene has recently been increasing.

The detection methods of these biosensors are broadly classified into optical energy transfer/conversion and electrical conversion methods depending on the type and method of the signal being measured. In particular, field-effect transistor (FET)-based biosensor devices that use one of the electrical conversion methods have advantages such as miniaturization, mass production, single cell and single molecule analysis, real-time observation, and low cost.

In addition, as biomarker technology has recently developed, electronic sensor technology that can detect and amplify electrical signals specified by biomarkers for inspection and diagnosis is becoming a reality. In addition, as part of establishing an at-home quarantine system, the demand for technology to diagnose diseases without visiting a hospital is increasing. In this regard, Republic of Korea Patent Publication No. 10-2022-0144284 relates to an ultra-fast and recyclable DNA biosensor for point-of-care detection of SARS-CoV-2, which is spaced apart from a glass substrate and an upper part of the glass substrate. Disclosed is a biosensor including a first electrode and a second electrode disposed, and probe DNA attached to the glass substrate exposed between the first electrode and the second electrode.

The purpose of the present invention is to provide an amino acid tag for immobilization on carbon nanomaterials and its use.

In order to achieve the above object, the present invention provides a tag for fixing to a carbon nanomaterial consisting of a polypeptide consisting of two or more identical amino acid residues, wherein the amino acid residues are phenylalanine, tryptophan, tyrosine, or histidine.

Additionally, the present invention provides a polynucleotide encoding the anchoring tag.

Additionally, the present invention provides an expression vector containing the polynucleotide operably linked to a protein of interest.

Furthermore, the present invention provides a composition and kit for producing a target protein that can be immobilized on a carbon nanomaterial containing the immobilization tag or expression vector.

The tag for fixing to carbon nanomaterials of the present invention is effectively fixed to carbon nanomaterials such as graphene without any other linker or chemical treatment, by fixing the target protein fused to its C-terminus to the carbon nanomaterial in a directionally oriented manner. , It can be usefully used in the manufacture of biosensors that convert biological signals into electrochemical signals.

Figure 1 is a graph showing the photo (A) and the fluorescence intensity quantified as a result of confirming the effect of fixing the streptoamicin protein-fused tag to the graphene sheet according to the type of amino acid residue, prepared in an example of the present invention ( B) is.

Figure 2 is a graph showing the photo (A) and the fluorescence intensity quantified as a result of confirming the fixation effect on the graphene sensor according to the type of amino acid residue of the streptoamicin protein-fused tag, prepared in an example of the present invention ( B) is.

Figure 3 is a photo (A) showing the results of confirming the fixation effect on the graphene sensor according to the number of amino acids of the tag fused with the scFv for the spike protein of SARS-CoV-2, prepared with tryptophan residue in an example of the present invention. and a graph (B) showing the quantification of fluorescence intensity.

Figure 4 confirms the effect of fixation on the graphene sheet according to the number of amino acids of the tag fused with the scFv for the spike protein of SARS-CoV-2, prepared with a tryptophan residue in an embodiment of the present invention, and shows the fluorescence intensity. This is a graph showing the quantified results.

Figure 5 is a graph showing the results of confirming changes in the function of the spike protein of SARS-CoV-2 due to a tag made from a tryptophan residue in one embodiment of the present invention.

Figure 6 is a graph showing the results of confirming the effect of a biosensor on which the spike protein of SARS-CoV-2 fused with a tag prepared with a tryptophan residue is immobilized in an embodiment of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention consists of a polypeptide consisting of two or more identical amino acid residues, wherein the amino acid residues are phenylalanine (Phe, F), tryptophan (Trp, W), tyrosine (Tyr, Y) or histidine (Hi, H). Provides a tag for fixing to carbon nanomaterials.

As used herein, the term "tag" refers to a peptide sequence that can be linked, attached, or fused to a target protein, and may be used in the same sense as protein tag, peptide tag, fusion tag, tag protein, or marker protein. You can. The tag may have a carbon nanomaterial connected to its N-terminus, and a target protein may be connected to its C-terminus.

The term "target protein" refers to a protein to be immobilized on a carbon nanomaterial, and can specifically bind to the protein to be detected and convert a biological signal into an electrochemical signal using the carbon nanomaterial. That is, the target protein may include both the protein to be detected and a protein that can bind through antigen-antibody binding, ligand-receptor binding, etc. For example, the target protein may include an antibody or antigen-binding fragment thereof, an antigen, a ligand protein, a receptor protein, etc. At this time, the antigen-binding fragment may refer to the portion excluding the Fc (crystallizable fragment) of an antibody that functions to transmit the stimulation of binding to the antigen to cells, complement, etc. For example, the antigen-binding fragment of an antibody may include Fab, scFv, F(ab) ₂ , and Fv, and may also include third-generation antibody fragments such as single domain antibodies or minibodies. You can.

The tag according to the present invention can be fused with the target protein as described above by a conventional method. For example, the tag can be synthesized at the N-terminus of the polypeptide sequence constituting the target protein by a conventional method. In addition, the tag can be expressed in a fused form to the target protein by inserting a polynucleotide sequence encoding the tag to be operably expressed at the 5-terminus of the polynucleotide sequence encoding the target protein.

The tag may be composed of two or more identical amino acid residues, specifically, the tag may have 2 to 20 amino acid residues, 2 to 16 amino acid residues, 2 to 13 amino acid residues, 2 to 10 amino acid residues, 2 to 7 amino acid residues, and 2 to 4 amino acid residues. , 5 to 20, 5 to 17, 5 to 14, 5 to 11, 5 to 8, 8 to 20, 8 to 17, 8 to 14, 8 to 11, 11 to 20 , 11 to 17, 11 to 14, 14 to 20, or 14 to 17 identical amino acid residues. For example, if the tag consists of two identical amino acid residues, the tag may be FF, WW, YY or HH, and if the tag consists of three identical amino acid residues, the tag may be FFF, WWW , YYY or HHH.

As used herein, the term “carbon nanomaterial” refers to a nanoscale conductive material made of hexagonal carbon, and can be used to make bendable electronic materials. The carbon nanomaterials are used in the manufacture of biosensors and may include all types of carbon nanomaterials known in the art. For example, the carbon nanomaterial may be a graphene material, carbon nanotube, carbon nanowire, or fullerene. At this time, the graphene material may be graphene nanoflake, graphene oxide (GO), reduced graphene (rGO), or CVD graphene (chemical vapor deposition graphene).

The tag according to the present invention can be modified with phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, etc., as needed.

Additionally, the present invention provides a polynucleotide encoding the anchoring tag.

The polynucleotide may be DNA or RNA encoding an anchoring tag having the characteristics described above. The sequence of the polynucleotide is obvious to those skilled in the art as long as the polypeptide sequence constituting the tag is known.

Additionally, the present invention provides an expression vector containing the polynucleotide operably linked to a protein of interest.

As used herein, the term 'expression vector' refers to a means for expressing a target gene in a host cell and may include plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, etc. The expression vector may contain elements necessary for producing a peptide from the nucleic acid contained therein. Specifically, the expression vector may include a signal sequence, origin of replication, marker gene, promoter, transcription termination sequence, etc. At this time, the polynucleotide according to the present invention can be operably linked to the target protein and promoter.

As used herein, “operably linked” means linked for translation in an expression cassette that functions as a unit for expressing an exogenous protein. For example, a target protein and a promoter operably linked to an anchoring tag according to the present invention can produce a polypeptide by translating the anchoring tag and the target protein by the promoter.

For example, an expression vector used in prokaryotic cells may include a promoter for transcription, a ribosome binding site for initiation of translation, and termination sequences for transcription and translation. Meanwhile, expression vectors used in eukaryotic cells may include a promoter and polyadenylation sequence derived from a mammal or a mammalian virus.

Additionally, any marker gene included in the expression vector can be any one known in the art, and specifically, it may be an antibiotic resistance gene. Specifically, the antibiotic resistance gene may be a gene showing resistance to antibiotics including ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, neomycin, tetracycline, etc.

The polynucleotide included in the expression vector may have the characteristics described above. At this time, the polynucleotide may be operably linked to the gene encoding the target protein. Specifically, the polynucleotide may be operably linked to the 5'-end of the gene encoding the target protein. The polynucleotide may encode the fixation tag according to the present invention.

The present invention provides a composition and kit for producing a target protein that can be immobilized on a carbon nanomaterial containing the immobilization tag or expression vector.

The immobilization tag or expression vector included in the composition and kit according to the present invention may have the characteristics described above. In addition, the kit can be manufactured by conventional manufacturing methods known to those skilled in the art, and may further include buffers, stabilizers, inactive proteins, etc., if necessary.

Hereinafter, the present invention will be described in detail through the following examples. However, the following examples are only for illustrating the present invention and are not intended to limit the present invention thereto. Anything that has substantially the same structure as the technical idea described in the claims of the present invention and achieves the same operation and effect is included in the technical scope of the present invention.

Example 1. Preparation of 3×histidine tag

A 3×histidine polypeptide with three histidines linked together was synthesized by a conventional method, and FITC was bound to its C-terminus to prepare a 3×histidine tag.

Example 2. Preparation of 3×phenylalanine tag

A 3×phenylalanine tag was prepared in the same manner as Example 1, except that phenylalanine was used instead of histidine.

Example 3. Preparation of 3×tyrosine tag

A 3×tyrosine tag was prepared in the same manner as Example 1, except that tyrosine was used instead of histidine.

Example 4. Preparation of 3×tryptophan tag

A 3×tryptophan tag was prepared in the same manner as Example 1, except that tryptophan was used instead of histidine.

Example 5. Preparation of 6×tryptophan tag

A 6×tryptophan tag (SEQ ID NO: 1) was prepared in the same manner as Example 4, except that a polypeptide with 6 tryptophans linked instead of a polypeptide with 3 tryptophans linked was used.

Example 6. Preparation of 9×tryptophan tag

A 9×tryptophan tag (SEQ ID NO: 2) was prepared in the same manner as Example 4, except that a polypeptide with 9 tryptophans linked instead of a polypeptide with 3 tryptophans linked was used.

Example 7. Preparation of 12×tryptophan tag

A 12×tryptophan tag (SEQ ID NO: 3) was prepared in the same manner as in Example 4, except that a polypeptide with 12 tryptophan links was used instead of a polypeptide with 3 tryptophan links.

Example 8. Preparation of 15×tryptophan tag

A 15×tryptophan tag (SEQ ID NO: 4) was prepared in the same manner as Example 4, except that a polypeptide with 15 tryptophans was used instead of a polypeptide with 3 tryptophans connected.

Experimental Example 1. Confirmation of fixation effect on graphene sheet according to amino acid type

The effect of fixing the produced amino acid tag to the graphene sheet according to the type of amino acid was confirmed as follows.

First, the amino acid tags of Examples 1 to 4 were prepared by diluting them in dimethyl sulfoxide (DMSO) to a concentration of 1 mM. Meanwhile, a graphene sheet was manufactured by applying graphene to a semiconductor wafer made of silicon and cutting it. The prepared graphene sheet was washed three times with 99.9% ethanol and three times with distilled water, and treated with the amino acid tag diluted in DMSO in an amount of 10 μl. This was reacted overnight at 4°C in the dark, and the amino acid tag was fixed to the graphene sheet. After the reaction was completed, the graphene sheet was washed three times with distilled water, and the amino acid tag fixed on the graphene sheet was confirmed using an upright fluorescence microscope. As a result, a picture taken with a fluorescence microscope is shown in Figure 1A, and a graph measuring the fluorescence intensity is shown in Figure 1B.

As shown in Figure 1, the amino acid tag was immobilized on the graphene sheet, and in particular, the tag prepared using tyrosine or tryptophan had a significantly better immobilization effect on the graphene sheet.

Experimental Example 2. Confirmation of fixation effect on graphene sensor according to amino acid type

The fixation effect of the produced amino acid tag on the graphene sensor according to the type of amino acid was confirmed as follows. The experiment was performed in the same manner as Experimental Example 1, except that a graphene sensor was used instead of a graphene sheet. At this time, the graphene sensor was manufactured by performing photoresist work on the graphene sheet manufactured as described above by a conventional method. As a result, the tag bound to the graphene sensor was photographed using a fluorescence microscope. A photo of the result is shown in Figure 2A, and a graph measuring the fluorescence intensity is shown in Figure 2B.

As shown in Figure 2, tags prepared using tyrosine or tryptophan were effectively immobilized on the graphene sensor.

Experimental Example 3. Confirmation of fixation effect on graphene sensor according to amino acid length

Through the above experimental example, the tryptophan tag, which was confirmed to be immobilized on graphene with the best binding force, was used to confirm the immobilization effect according to the amino acid length. The experiment was performed in the same manner as Experiment 2 above, except that the amino acid tags prepared in Examples 4 to 6 were used. As a result, the tag bound to the graphene sensor was photographed using a fluorescence microscope. A photo of the result is shown in Figure 3A, and a graph measuring the fluorescence intensity is shown in Figure 3B.

As shown in Figure 3, the fixation effect on the graphene sensor was significantly increased in the tag manufactured by connecting six or more tryptophans.

Experimental Example 4. Optimization of amino acid length

The fixation effect with the graphene sheet according to the number of tryptophan was confirmed in the following manner.

First, a polynucleotide encoding 3, 6, or 9 tryptophan tags was inserted into the pET-42 vector to express streptomycin protein at its C-terminus. The recombinant pET-42 vector was transformed into E. coli, and recombinant E. coli expressing streptomycin with a tryptophan tag was selected. The selected E. coli were cultured at 37°C at a speed of 150 rpm using LB culture medium to a total volume of 6 liters using six 2 liter flasks. When the OD ₆₀₀ reached 0.45 to 0.5, 1 ml of 1 M IPTG was added to each flask and incubated for another 4 hours under the same conditions. After culturing, the cells were recovered by centrifugation, and histidine binding buffer (20mM Tris-HCl, 0.5M NaCl, 20mM imidazole, pH 8.0) was added to the cells and vortexed. The cells were lysed by sonication, and centrifuged again to obtain the supernatant. The obtained supernatant was filtered through a filter with a pore size of 0.4 ㎛, and his-tag affinity chromatography was performed in a conventional manner to obtain the streptomycin protein bound to the tryptophan tag. was purified. Afterwards, a graphene sheet was attached to the bottom of a 96-well plate, and 200 ㎕ of the purified streptomycin protein was treated. After storing it at 4°C overnight, the wells were washed three times using PBS. BSA solution was added thereto and pretreated for 1 hour, and the fixation effect of the tryptophan tag on the graphene sheet was confirmed through fluorescence using a primary antibody against HRP-conjugated streptavidin. As a result, a graph measuring the fluorescence intensity is shown in Figure 4.

As shown in Figure 4, the fixation effect on the graphene sheet was significantly increased in the tag manufactured by connecting six or more tryptophans.

Experimental Example 5. Confirmation of effect of amino acid tag on antibody binding ability

Whether there was a change in the binding affinity of the amino acid tag-conjugated antibody to the target protein was confirmed using the following method.

Specifically, a polynucleotide encoding 3, 6, 9, 12 or 15 tryptophan tags is added to its C-terminus as an scFv (SEQ ID NO: 5) that specifically binds to the spike protein of SARS-CoV-2. ) was inserted into the pET-42 vector to express the gene. The pET-42 vector into which the scFv gene was inserted was transformed into E. coli as described above and purified using a Ni column by a conventional method. 100 μl of the purified protein (1 μM) was added to a 96-well plate with a graphene sheet attached to the bottom, and left at room temperature for 2 hours. Afterwards, the plate was washed three times with PBS buffer and pretreated with 100 μl of 2% BSA buffer for 1 hour at room temperature. This was washed again three times with PBS buffer, and 100 μl of SARS-CoV-2 spike protein (SEQ ID NO: 6) was added to each well. After being left at room temperature for 1 hour, it was washed three times with PBS buffer and treated with a primary antibody against the spike protein of SARS-CoV-2 bound to HRP at a dilution of 1:2000. After reacting at room temperature for 1 hour, the mixture was washed four times with PBS buffer, 50 ㎕ of TMB solution was added, light was blocked, and reaction was allowed at room temperature for 20 minutes. After the reaction was completed, 50 μl of 2 M sulfuric acid solution was added to stop the reaction, and the absorbance was measured at a wavelength of 450 nm, and the results are shown in Figure 5.

As shown in Figure 5, the scFv fused with the tryptophan tag was immobilized on the graphene sheet with excellent anchoring force, and the effect was significantly increased in the tag prepared by connecting 6 or more tryptophans.

Experimental Example 6. Confirmation of graphene sensor response

10 ㎕ of scFv fused with the tryptophan tag purified in Experimental Example 5 was applied to the graphene sensor and left at room temperature for 2 hours. Afterwards, the sensor was washed three times with PBS buffer and treated with 1 μl of SARS-CoV-2 protein. The probe was grounded to the source, drain, and gate electrodes of the protein-treated graphene sensor, and the current was measured using a source meter. As a result, the measured current signal is shown in Figure 6.

As shown in Figure 6, it was found that the spike protein of SARS-CoV-2 could be detected as a biosensor by scFv fused with a tryptophan tag.

Claims (11)

1. Consists of a polypeptide consisting of two or more identical amino acid residues,

   A tag for fixing to a carbon nanomaterial wherein the amino acid residue is phenylalanine, tryptophan, tyrosine, or histidine.
2. The tag for fixing to a carbon nanomaterial according to claim 1, wherein the number of amino acid residues is 2 to 20.
3. The tag for fixing to a carbon nanomaterial according to claim 1, wherein the tag is connected to a carbon nanomaterial at its N-terminus.
4. The tag for fixing to a carbon nanomaterial according to claim 1, wherein a target protein is linked to the C-terminus of the tag.
5. The tag for fixing to a carbon nanomaterial according to claim 4, wherein the target protein is an antibody, an antigen-binding fragment thereof, an antigen, a ligand protein, or a receptor protein.
6. The tag for fixing to a carbon nanomaterial according to claim 1, wherein the carbon nanomaterial is a graphene material, carbon nanotube, carbon nanowire, or fullerene.
7. A polynucleotide encoding the anchoring tag of claim 1.
8. An expression vector comprising the polynucleotide of claim 7 operably linked to a gene encoding a target protein.
9. The expression vector according to claim 8, which is linked to the 5'-end of the gene encoding the target protein.
10. A composition for producing a target protein that can be immobilized on a carbon nanomaterial, comprising the immobilization tag of claim 1 or the expression vector of claim 8.
11. A kit for producing a target protein that can be immobilized on a carbon nanomaterial containing the immobilization tag of claim 1 or the expression vector of claim 8.

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