KR101451229B1 - Method for protein labeling method using unnatural amino acid and protein labeling composition thereof - Google Patents

Abstract

A method for labeling a protein including an unnatural amino acid, a method for analyzing a labeled protein, and a composition for protein labeling

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a protein labeling method using an unnatural amino acid, and a protein labeling method therefor. 2. The protein labeling method according to claim 1,

The present invention relates to a labeling method for a protein containing an unnatural amino acid, a method for analyzing a labeled protein, and a composition for protein labeling.

Numerous techniques for labeling target proteins using biochemical probes have been developed to study the structure and function of proteins. However, these methods have been limited in their utility due to their low efficiency, slow reaction rate, biologically incompatible reaction conditions, large size of label groups, and the need for specific side chains such as cysteine or lysine.

Alternative methods include labeling the target protein with a peptide / protein tag such as a fluorescent protein, SNAP-tag, HaloTag, or tetracysteine. For example, Korean Patent Publication No. 2009-0100407 discloses compositions containing unnatural amino acids and polypeptides, methods involving unnatural amino acids and polypeptides, and uses of unnatural amino acids and polypeptides. Although these tagging methods are particularly useful for in vivo applications, most of these tags can disturb the structure and function of the tagged target protein due to their large size. In addition, these tags have limitations in their application because of their limitations that can only be located at the N- or C-terminus of the target protein.

To overcome these limitations, non-natural amino acids with bio-orthogonal reactivity have been introduced into target proteins using recently developed genetic induction methods. A major advantage of this method is that it is possible to carry out site-specific protein conjugation by biochemical probes. In addition, this method is technically simple and applicable to all proteins. Bioisogonal functional groups that can be introduced into the target protein by the genetic methods include ketones, azides, alkanes, and alkenes.

Although the site-specific introduction of these functional groups into the target protein is quite useful, the applicability of this method is limited by the stringent reaction conditions of the conjugation reaction and the slower reaction rate. The imine formation reaction between the ketone and the alkoxyamine requires acidic conditions (pH 4 to 5), and the click reaction involving the azide and alkane requires a copper (I) catalyst that is incompatible with the living body. In recent years, it has been reported that the cyanobenzothiazole condensation with 1,2-aminothiol, and the tetrazine-based norbornene-containing Such as the addition of cyclization to proteins, have been introduced but still have limitations due to the low conjugation rate, slow reaction rate, the need for additional processing steps, and the difficulty of synthesizing the labeled compound.

Therefore, there is a need for an improved technique for efficiently labeling a target protein by simply mixing a protein of interest and a labeling reagent under physiological affinity conditions.

In addition, the Western blot analysis method, which is one of powerful analytical techniques for searching for a specific protein, has a very high specificity and sensitivity, but an antibody specifically binding to a target protein is essential for analysis, A washing step is required. Therefore, it is expected that simple and non-antibody-requiring protein assay methods with high specificity and sensitivity will have unlimited potential and wide applicability.

The present invention relates to a process for the preparation of a target protein by conjugation reaction between the azide group of a target protein comprising an azide group-containing unnatural amino acid and a cycloalkynyl group bonded to the dye, A method for analyzing a protein, a method for analyzing a protein, and a composition for labeling a protein, wherein the target protein is directly labeled to allow separation, purification, and Western blotting.

However, the problems to be solved by the present invention are not limited to the above-mentioned problems, and other problems not mentioned can be clearly understood by those skilled in the art from the following description.

According to a first aspect of the present invention, there is provided a method for amplifying a target protein comprising the steps of: substituting an amino acid contained in a target protein with an azide group-containing unnatural amino acid; And labeling the target protein with the labeling reagent through a conjugation reaction between the cycloalkynyl group and the azide group contained in a labeling reagent comprising a cycloalkynyl group, , And provides a labeling method for proteins.

The second aspect of the present invention provides a method for analyzing a protein, which comprises purifying a target protein labeled by the labeling method of the protein according to the first aspect of the present application.

A third aspect of the present invention is a labeling reagent comprising a labeling reagent formed by reacting a dye and a cycloalkynyl group-containing compound, wherein the cycloalkynyl group contained in the labeling reagent and an azide group- There is provided a composition for labeling a protein, wherein the azide group of a target protein containing a natural amino acid is used to label the target protein by causing a conjugation reaction.

According to the present invention, there is provided a labeling method of a protein capable of labeling a target protein with high efficiency under physiological conditions by simply mixing a target protein and a labeling reagent, a method for analyzing a protein using the same, and a composition for purifying a protein can do. The labeling method according to the present invention is simple and biocompatible since it does not require antibody or multistage incubation and does not require a copper (Cu) catalyst which is toxic to the living body in the labeling reaction. In addition, since the labeling method of the present invention is very specific, very small amounts of target proteins can be labeled and high purity purification and quantitative analysis are possible. In addition, the labeled proteins according to the present invention can be separated and purified by the anti-antibody western blot, and can be very useful for labeling and analyzing proteins.

1 is a schematic diagram of a labeling reagent synthesis process according to an embodiment of the present invention.
Figure 2 is a schematic diagram of a strain-facilitated azide-alkane cycloaddition (SPAAC) reaction according to one embodiment of the present disclosure.
FIG. 3 shows SDS-PAGE analysis (FIG. 3A) and MALDI-TOF analysis (FIG. 3A) showing the expression of glutathione-S-transferase (GST) containing an azide group-containing unnatural amino acid according to an embodiment of the present invention 3B).
4A to 4C are graphs showing the results of an analysis showing that the glutathione-S-transferase containing an azide group-containing unnatural amino acid according to an embodiment of the present invention was labeled with a labeling reagent Cy5.5-ADIBO to be.
FIG. 5 shows labeling and analysis results of the glutathione-S-transferase containing the azide group-containing unnatural amino acid contained in the cell-cell extract according to one embodiment of the present invention.
FIG. 6 is a fluorescence image and a Coomassie staining image for analysis of the conjugation result of GST containing CyAphenylalanine and Cy5.5-ADIBO according to one embodiment of the present invention. FIG.
FIG. 7 is a graph showing the results of quantitative analysis of expression amounts of GST containing azido phenylalanine labeled with Cy5.5-ADIBO by western blot according to one embodiment of the present invention in a Coomassie staining image and a fluorescence image will be.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily carry out the present invention. It should be understood, however, that the present invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. In the drawings, the same reference numbers are used throughout the specification to refer to the same or like parts.

Throughout this specification, when a part is referred to as being "connected" to another part, it is not limited to a case where it is "directly connected" but also includes the case where it is "electrically connected" do.

Throughout this specification, when a member is "on " another member, it includes not only when the member is in contact with the other member, but also when there is another member between the two members.

Throughout this specification, when an element is referred to as "including " an element, it is understood that the element may include other elements as well, without departing from the other elements unless specifically stated otherwise. The terms "about "," substantially ", etc. used to the extent that they are used throughout the specification are intended to be taken to mean the approximation of the manufacturing and material tolerances inherent in the stated sense, Accurate or absolute numbers are used to help prevent unauthorized exploitation by unauthorized intruders of the referenced disclosure. The word " step (or step) "or" step "used to the extent that it is used throughout the specification does not mean" step for.

Throughout this specification, the term "combination thereof" included in the expression of the machine form means one or more combinations or combinations selected from the group consisting of the constituents described in the expression of the machine form, And the like.

Throughout this specification, "unnatural amino acid" refers to an amino acid that is not one of the 20 common amino acids, pyrosinine, or selenocysteine; Other terms that may be used in a similar sense to the term " unnatural amino acid "include" non-cirrhically coded amino acids "and" naturally occurring amino acids ". An "unnatural amino acid" includes, but is not limited to, an amino acid that occurs naturally by modification of a naturally encoded amino acid, but which is not itself introduced into a polypeptide grown by a translation complex. Such naturally encoded amino acids include, but are not limited to, 20 common amino acids, pyrosinine, or selenocysteine.

Hereinafter, embodiments and examples of the present invention will be described in detail with reference to the accompanying drawings, but the present invention is not limited thereto.

According to a first aspect of the present invention, there is provided a method for amplifying a target protein comprising the steps of: substituting an amino acid contained in a target protein with an azide group-containing unnatural amino acid; And labeling the target protein with the labeling reagent through a conjugation reaction between the cycloalkynyl group and the azide group contained in a labeling reagent comprising a cycloalkynyl group, , A method of labeling a protein can be provided.

For example, the labeling reagent may include a dye. For example, the azido group-containing unnatural amino acid may be introduced by a genetic method, but is not limited thereto.

For example, the nucleic acid sequence encoding a target protein comprising the unnatural amino acid is based on the amino acid sequence of a wild-type protein, but in order to introduce, substitute, or remove a specific amino acid contained in the amino acid sequence, The nucleic acid sequence corresponding to the amino acid sequence can be changed, but is not limited thereto. The nucleic acid sequence may be optionally or nonselectively modified according to methods known in the art. For example, a codon encoding a specific amino acid in the gene of the protein may be substituted with a stop codon including an amber codon, an opal codon, or an ocher codon; A plasmid containing a gene of a protein having a codon substituted with the stop codon, a plasmid containing a suitable tRNA gene, and a plasmid containing a suitable aminoacyl tRNA synthetase gene are introduced into a cell, for example, Escherichia coli, Producing introduced cells; And culturing the transduced cells in a medium containing the unnatural amino acid, but the present invention is not limited thereto.

For example, when the transduced cell produces a protein mutant, the unnatural amino acid may bind to the tRNA by the aminoacyl tRNA synthetase, and the tRNA bound to the unnatural amino acid may be substituted But is not limited to, recognizing the nucleic acid sequence and inserting the unnatural amino acid into the appropriate site of the protein to be translated. The unnatural amino acid may be at any position on the protein, including any terminal position or any internal position of the protein. The non-natural amino acid-introduced protein may be produced biosynthetically, but is not limited thereto. The term "biosynthetically" as used herein refers to a method using a cellular translation system, including, but not limited to, using one or more of polynucleotides, codons, tRNAs, and ribosomes.

For example, the position of the amino acid that may be substituted with the unnatural amino acid may be a position excluded from a potential receptor binding site that is a site that binds to one or more binding partners, a position that can be completely or partially exposed to a solvent, As expected from a minimal or no hydrogen bonding interaction with the residue, minimal exposure to proximal reactive moieties, a three-dimensional, secondary, tertiary, or quaternary structure of the protein, A position that is at a site that is structurally rigid, that binds to or does not bind to a related receptor, ligand or binding protein, that binds to another protein or other biologically active molecule, or that does not bind to it, To include the protein itself, or one or more proteins Including a position that can be adjusted to three-dimensional form of the dimer or oligomer is not one that is, limited.

Because the unnatural amino acids herein are typically different from the native amino acids in only the side chain structure, the unnatural amino acids are formed from other amino acids and naturally occurring proteins including, but not limited to, natural or non-naturally encoded The amide bond can be formed in the same manner as in the case of the amide bond.

For example, the azido group-containing unnatural amino acid can be introduced genetically into all kinds of proteins. This means that it is possible to replace the nucleic acid sequence corresponding to one or more amino acids contained in the protein with another nucleic acid sequence and thereby replace the amino acid contained in the protein with another amino acid regardless of the kind of the target protein, Because it is extremely easy for a person with ordinary knowledge to perform it.

For example, the target protein may be selected from the group consisting of glutathione-S-transferase, insulin, growth hormone, interferon-gamma, hematopoietic growth factor, bone morphogenetic protein, renin, growth hormone releasing factor, Hormone, thyroid stimulating hormone, lipoprotein, alpha-1-antitrypsin, insulin A-chain, insulin B-chain, proinsulin, follicle stimulating hormone, calcitonin, luteinizing hormone, glucagon, factor VIIIC, factor IX, (T-PA), bombesin, thrombin, tumor necrosis factor-alpha and tumor necrosis factor (TNF-alpha), tumor necrosis factor (S) selected from the group consisting of: - beta, Encepinalinase, RANTES, human macrophage inflammatory protein (MIP-1-alpha), serum albumin, mullerian-inhibitory substance, lilacsin A-chain, lilacsin B- (CTLA), inhivin, actibin, vascular endothelial growth factor (VEGF), receptors of hormone or growth factor, protein A or D, cytotoxic T-lymphocyte associated antigen (BDNF), neurotrophin-3, -4, -5 or -6, nerve growth factor, platelet derived growth factor (PDGF), fibroblast growth factor, epidermal growth factor (EGF) , IGF-I, insulin-like growth factor binding protein (IGF-BP), CD protein, erythropoietin, bone induction, Interferon beta, colony stimulating factor (CSF), interleukin 1 to interleukin 10, superoxide dismutase, T-cell receptor, surface membrane protein, collapse-promoting factor, viral antigen, transport protein, Homing receptors, adrencine, regulatory proteins, integrins Lin, tumor associated antigens, however, and may be selected from the group consisting of fragments thereof, without being limited thereto.

According to one embodiment of the present invention, the labeling reagent may include, but is not limited to, those formed by reacting a dye with a cycloalkynyl group-containing compound.

For example, the cycloalkynyl group-containing compound may contain one, two, or three or more phenyl groups, but is not limited thereto.

According to one embodiment of the disclosure, the cycloalkynyl group may contain heteroatoms, but is not limited thereto. For example, the hetero atom may include, but is not limited to, an atom selected from the group consisting of a nitrogen atom, an oxygen atom, a sulfur atom, and combinations thereof. For example, the cycloalkynyl group may contain one heteroatom, two heteroatoms, three heteroatoms, or four heteroatoms, but is not limited thereto.

In one embodiment of the present invention, the cycloalkynyl group includes a cyclobutynyl group, a cyclopentinyl group, a cyclohexynyl group, a cycloheptynyl group, a cyclooctynyl group, a cyclononynyl group, or a cyclodecynyl group , But is not limited thereto.

In one embodiment of the present invention, the cycloalkynyl group may be, but not limited to, a dibenzocyclooctinyl group or a dibenzocyclooctane derivative.

For example, the cyclooctinyl group may include, but is not limited to, a dibenzocyclooctinyl group or a dibenzocyclooctane derivative.

For example, the cyclooctynyl group may be included in aza-dibenzocyclooctyne (ADIBO), but is not limited thereto.

According to one embodiment of the invention, the dye may include, but is not limited to, a radioactive dye, a fluorescent dye, and a biological dye.

According to one embodiment of the present invention, the fluorescent dye may include, but is not limited to, cyanine, rhodamine, coumarine, BODIPY, or fluorescein It is not. For example, the fluorescent dye may include Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, or Cy7, but is not limited thereto.

According to an embodiment of the present invention, the labeling reagent may include Cy5.5-linked aza-dibenzocyclooctyne (Cy5.5-ADIBO) It is not.

For example, the combination Cy5.5- aza-dibenzo-cyclooctadiene in the amide bond between the can ADIBO-NH ₂ and N- hydroxy-Cy5.5 as disclosed in one shinny imide ester (Cy5.5 NHS ester) But the present invention is not limited thereto.

According to one embodiment herein, the azido group-containing unnatural amino acid is azidophenylalanine (AZF) or N6 - [(2-azidethoxy) carbonyl] -l-lysine [ (2-azidoethoxy) carbonyl] -l-lysine], but the present invention is not limited thereto.

According to one embodiment of the present application, the conjugation reaction is carried out by strain-promoted azide-alkyne cycloaddition (SPAAC) between the cycloalkynyl group and the azide group But is not limited thereto.

The SPAAC is a reaction having high sensitivity and specificity, and Fig. 2 is a schematic diagram showing the process of SPAAC. As shown in FIG. 2, the azide group contained in the target protein into which the azide group-containing unnatural amino acid is genetically introduced causes a conjugation reaction with dibenzocyclooctane and SPAAC.

SPAAC with dibenzocyclooctane derivatives is well known to be ideal for bioconjugation in living organisms because it does not require additional reagents and has a high rate constant (0.1-1.0 M ^-1 s ^-1 ). Because of these advantages, SPAAC has been used in a variety of processes, including labeling or functionalizing cell surfaces. To apply SPAAC to protein conjugation, an azide group must be introduced into the target protein as a conjugation. The introduction of azide groups into the target protein can be achieved by genetic methods, so application of SPAAC to genetically introduced azide group-containing amino acids can enable effective labeling of the target protein.

According to one embodiment of the present invention, the conjugation reaction may be initiated by mixing the target protein with the labeling reagent, but is not limited thereto. According to the labeling method of the present invention, the target protein can be analyzed by an analytical method such as SDS-PAGE by mixing with a labeling reagent, but the present invention is not limited thereto.

According to one embodiment of the present invention, the target protein may include, but is not limited to, a glutathione-S-transferase (GST).

For example, the labeling method of the present protein may be capable of labeling a target protein with a labeling ratio of about 80% within about 120 minutes, but is not limited thereto. For example, the labeling method of the present protein may comprise from about 70% to about 95%, from about 75% to about 95%, from about 80% to about 95%, from about 85% to about 95%, from about 90% To about 95%, about 70% to about 90%, about 70% to about 85%, about 70% to about 80%, or about 70% to about 75% But is not limited thereto. In addition, quantitative analysis of target protein expression can be performed using the labeling method of the present invention, but the present invention is not limited thereto. In addition, the labeling methods of the present proteins can be used to perform, but not limited to, SDS-PAGE without antibody, multistage incubation, and / or washing steps.

The second aspect of the present application can provide a method for analyzing a protein, which comprises purifying a target protein labeled by the labeling method of the protein according to the first aspect of the present invention.

In one embodiment of the present invention, the method for analyzing the protein may further include quantitative analysis or qualitative analysis of the purified target protein, but the present invention is not limited thereto.

For example, the method of analyzing the protein includes electrophoresis, Western blot, ELISA (enzyme-linked immunosorbent assay), or chromatography including SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) But is not limited thereto.

For example, the method of analyzing the protein may include, but is not limited to, an anti-antibody western blot.

A third aspect of the present invention is a labeling reagent comprising a labeling reagent formed by reacting a dye and a cycloalkynyl group-containing compound, wherein the cycloalkynyl group contained in the labeling reagent and an azide group- It is possible to provide a composition for labeling a protein, which is used for labeling the target protein by causing a conjugation reaction of the azide group of a target protein containing a natural amino acid.

For example, the azido group-containing unnatural amino acid can be introduced genetically into all kinds of proteins. This is because, regardless of the kind of protein, substitution of the nucleic acid sequence corresponding to one or more amino acids contained in the protein with another nucleic acid sequence and substitution of the amino acid contained in the protein with another amino acid is not required in the art This is because a person with ordinary knowledge can easily think or perform it.

For example, the target protein may be selected from the group consisting of glutathione-S-transferase, insulin, growth hormone, interferon-gamma, hematopoietic growth factor, bone morphogenetic protein, renin, growth hormone releasing factor, Hormone, thyroid stimulating hormone, lipoprotein, alpha-1-antitrypsin, insulin A-chain, insulin B-chain, proinsulin, follicle stimulating hormone, calcitonin, luteinizing hormone, glucagon, factor VIIIC, factor IX, (T-PA), bombesin, thrombin, tumor necrosis factor-alpha and tumor necrosis factor (TNF-alpha), tumor necrosis factor (S) selected from the group consisting of: - beta, Encepinalinase, RANTES, human macrophage inflammatory protein (MIP-1-alpha), serum albumin, mullerian-inhibitory substance, lilacsin A-chain, lilacsin B- (CTLA), inhivin, actibin, vascular endothelial growth factor (VEGF), receptors of hormone or growth factor, protein A or D, cytotoxic T-lymphocyte associated antigen (BDNF), neurotrophin-3, -4, -5 or -6, nerve growth factor, platelet derived growth factor (PDGF), fibroblast growth factor, epidermal growth factor (EGF) , IGF-I, insulin-like growth factor binding protein (IGF-BP), CD protein, erythropoietin, bone induction, Interferon beta, colony stimulating factor (CSF), interleukin 1 to interleukin 10, superoxide dismutase, T-cell receptor, surface membrane protein, collapse-promoting factor, viral antigen, transport protein, Homing receptors, adrencine, regulatory proteins, integrins Lin, tumor associated antigens, however, and may be selected from the group consisting of fragments thereof, without being limited thereto.

According to one embodiment of the disclosure, the cycloalkynyl group may contain heteroatoms, but is not limited thereto. For example, the hetero atom may include, but is not limited to, an atom selected from the group consisting of a nitrogen atom, an oxygen atom, a sulfur atom, and combinations thereof. For example, the cycloalkynyl group may be, but is not limited to, a heteroatom, two heteroatoms, or a cyclooctynyl group containing three heteroatoms.

According to one embodiment of the present invention, the cycloalkynyl group may be a cyclobutynyl group, a cyclopentinyl group, a cyclohexynyl group, a cycloheptinyl group, a cyclooctynyl group, a cyclononynyl group, or a cyclodecynyl group But are not limited thereto.

In one embodiment of the present invention, the cyclooctynyl group-containing compound may include, but is not limited to, dibenzocyclooctane, or dibenzocyclooctane derivatives.

According to an embodiment of the present invention, the labeling reagent may include Cy5.5-linked aza-dibenzocyclooctyne (Cy5.5-ADIBO) It is not. For example, the cyclooctynyl group may be contained in the Cy5.5-linked aza-dibenzocyclooctane, but is not limited thereto.

According to one embodiment of the present application, the conjugation reaction is carried out by strain-promoted azide-alkyne cycloaddition (SPAAC) between the cycloalkynyl group and the azide group But is not limited thereto.

According to one embodiment of the invention, the dye may include, but is not limited to, a radioactive dye, a fluorescent dye, and a biological dye. For example, the fluorescent dye may include, but is not limited to, cyanine, rhodamine, coumarine, BODIPY, or fluorescein. For example, the fluorescent dye may include Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, or Cy7, but is not limited thereto.

According to one embodiment of the present invention, the azide group-containing unnatural amino acid may be azido phenylalanine or N6 - [(2-azidethoxy) carbonyl] -l-lysine, It is not.

According to one embodiment of the present invention, the target protein may include, but is not limited to, a glutathione-S-transferase (GST).

According to one embodiment of the invention, the dye may include, but is not limited to, a radioactive dye, a fluorescent dye, and a biological dye.

Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited thereto.

[ Example ]

In this example, all chemicals and DNA oligomers were used without further purification, except those specifically mentioned.

< Cy5 .5-bond Keep it up - dibenzocyclooctane ( Cy5 .5- linked aza -dibenzocyclooctyne, Cy5 .5- ADIBO )>

To synthesize Cy5.5-linked aza-dibenzocyclooctane (Cy5.5-ADIBO), an aza-dibenzocyclooctynyl amine derivative, ADIBO-NH2 in anhydrous DMF (300 μL) ₂ Diisopropylethylamine (0.45 mg, 3.6 μmol) was added to a mixture of the above compound (4.0 mg, 13.8 μmol) and Cy5.5 N-hydroxy succinimide ester [Cy5.5 NHS-ester (2.0 mg, 1.8 μmol) Was added. Then, the reaction mixture was stirred at room temperature for 1 hour.

Reversed phase chromatography [ _C18 silica gel, 10 [mu] m, 10x250 mm; yield 2%) was obtained to obtain Cy5.5-ADIBO (2.0 mg, 93%) from the crude product thus obtained. _{10 mM aqueous NH 4 HCO 3 /} acetonitrile = 70:30 (v / v); 254 nm; 2 mL / min] was performed. The resulting Cy5.5-ADIBO product was identified by the following MALDI-TOF-MS: m / z 1211.08 for [M-3Na] (C ₆₂ H ₅₉ N ₄ O ₁₄ S ₄ calculated molecular weight [MW] 1211.29) . Retention time = 16.4 min.

< Phenylalanine  ( azidophenylalaine , AZF ) &Lt; / RTI &gt; Glutachi On-S-transferase ( GST ) &Lt; / RTI &gt;

The wildtype (WT) gene of glutathione-S-transferase (GST) was amplified by a commercial vector containing the GST gene and the GST gene was amplified by pBAD / Myc-His vector (Invitrogen) Was inserted at the site between NcoI and KpnI to generate pBAD-GST. In addition, an amber mutation (F47TAG) was introduced into the GST gene to generate a site-specific mutation to insert the unnatural amino acid (azido phenylalanine, AZF) into the 47th amino acid of the GST protein. The amber mutant plasmid (pBAD-GST-F47TAG) co-transfected into E. coli DH10B cells together with pEvol-AZF, a plasmid for protein introduction of AZF.

The cells were amplified in LB (lysogeny broth) medium containing ampicillin (100 μg / mL) and chloramphenicol (35 μg / mL). The cells were then seeded by inoculation (2.5 mL) at a temperature of 37 DEG C on 100 mL of LB medium containing ampicillin (100 mu g / mL), chloramphenicol (35 mu g / mL), and AZF 1 mM , And L-arabinose (0.2%, final concentration) were added at OD 0.8 (550 nm) to induce gene expression. The cells were incubated overnight at 37 ° C, harvested by centrifugation and frozen at -80 ° C. Subsequent GST resin purification was performed according to the manufacturer's protocol (Clontech Laboratories, Inc.), and the concentration of the GST protein was measured at 280 nm by calculating the extinction coefficient (4.3 × 10 ⁴ cm ^-1 M ^-1 ) Respectively.

<Refining GST Wow Cy5 .5- ADIBO of Conjugation  reaction( conjugation )>

The conjugation reaction between purified GST and Cy5.5-ADIBO was carried out by incubating mutant (GST (SEQ ID NO: 2)) inserted with AZF in the 47th amino acid of the GST protein in a 10 mM phosphate buffer solution (pH 7.0) containing 100 mM NaCl at room temperature, -F47AZF) 20 [mu] M (final concentration), and Cy5.5-ADIBO 200 [mu] M (final concentration). The reaction was stopped by adding an excess of AZF and the mixture was immediately analyzed by SDS-PAGE. Fluorescence images were obtained using a Typhoon 9210 variable mode imager and the gels were stained with Coomassie Brilliant Blue R-250.

<Cell Crude cell extract and Cy5 .5- ADIBO of Conjugation  Reaction>

From the bacterial culture expressing GST-F47AZF, 50 mL of the bacterial cells were collected by centrifugation and frozen at -80 ° C. The frozen cell pellet was thawed on ice and suspended in 10 mM phosphate buffer (pH 7.0, 5 mL) containing 100 mM NaCl and then sonicated. Alternatively, the thawed cells were suspended in BugBuster (Novagen) containing 10 mM phosphate buffer (pH 7.0), 100 mM NaCl, and benzoin (125 U / mL, cat. No. 70746-3, Novagen) And incubated at room temperature for 30 minutes. The suspended cells were centrifuged at 12,000 rpm to remove cell debris, and a cell crude extract was obtained.

The conjugation reaction was performed by adding 200 [mu] M Cy5.5-ADIBO (final concentration) to 10 [mu] L of cell extract and incubating the reaction mixture at 37 [deg.] C for 1 hour. The reaction was stopped by adding an excess of AZF and the mixture was immediately analyzed by SDS-PAGE.

<For protein expression level analysis Conjugation  reaction ( No antibody Western Blot  Analysis)>

Cells were cultured by the same method as described above, but different concentrations of L-arabinose (see FIG. 7). Preparation of cell lysates, conjugation reaction, and SDS-PAGE analysis were carried out in the same manner as described above.

< AZF of GST  Introduction and identification into>

As an unnatural amino acid for introducing an azide group into the target protein, p-azidophenylalanine (AZF) was selected as an azide group-containing amino acid and glutathione-S-transferase (GST) Was selected. Based on the X-ray crystal structure of GST, the 47th phenylalanine, the residue exposed to the solvent, was chosen as the residue to be replaced by AZF. For the introduction of the unnatural amino acid AZF, the codon corresponding to F47 was replaced with an amber codon (TAG) by site-directed mutagenesis.

The GST mutant protein (GST-F47AZF) containing AZF was expressed in E. coli DH10B cultivar cultured in LB medium in the presence of the corresponding tRNA / aminoacyl tRNA synthetase (aaRS) and 1 mM AZF. The mutant protein and wild-type protein (GST-WT) were purified by GST-affinity purification, and the yields of GST-F47AZF and GST-WT were 10-15 mg / L and 15-20 mg / L, respectively.

Additional SDS-PAGE analysis showed that when AZF was absent, small amounts of full length proteins were found by background incorporation (Fig. 3A). On the other hand, according to the MALDI-TOF mass spectrometry (MS) of FIG. 3B, normal proteins were not found in the purified proteins expressed in the presence of AZF, and AZF was introduced. The estimated mass difference between wild type and mutant proteins was 41 Da, and the measured mass difference was 37 Da.

<Refined GST -F47AZF and Cy5 .5- ADIBO Strain-facilitated Azid - Alkane  Cyclization addition ( SPAAC )On by Conjugation  Results Analysis>

In this example, a conjugation reaction of GST-F47AZF with a Cy5.5-linked aza-dibenzocyclooctane derivative (Cy5.5-linked aza-dibenzocyclooctyne derivative, Cy5.5-ADIBO) was performed. Cy5.5-ADIBO is ADIBO-NH was prepared from _2, ADIBO-NH ₂ and Cy5.5 N- hydroxy may form an amide bond between the shinny imide ester (Cy5.5 NHS ester) reaction is one in DMF at room temperature And then purified by high-performance liquid chromatography (HPLC) to obtain a bioorthogonal azide acceptor at a yield of 93%. Subsequently, the conjugation reaction was carried out using 10 μL of a reaction mixture containing 20 mM mutant GST and 200 μM Cy5.5-ADIBO in 100 mM phosphate buffer (pH 7.0) at room temperature and 100 mM NaCl. The conjugation reaction was analyzed by SDS-PAGE at different reaction times (Figures 4a and 4b).

Fluorescent images of SDS-PAGE showed that Cy5.5-ADIBO was efficiently conjugated to the mutant GST. As shown in the fluorescence image of FIG. 4A and the Coomassie stained image of FIG. 4B, fluorescence was not observed as a result of SDS-PAGE in the control group which had been subjected to the same conjugation reaction for 120 minutes using wild-type GST, The fluorescence observed in the reaction was the result of the specific conjugation of Cy5.5-ADIBO with the azido group introduced into the target protein. The conjugation reaction was also assessed by MALDI-TOF mass spectrometry performed on products that were reacted with the mutant GST for 120 minutes (Figure 4c). The mass difference between the predicted mutant GST and the conjugation protein was 1190 Da, and the measured mass difference was 1206 Da. The mass spectrometry results showed that a specific conjugation reaction took place and showed that approximately 80% of the mutated GSTs were conjugated.

This result shows that the AZF-containing protein can undergo a site-specific conjugation reaction within 120 minutes by simple mixing of the mutant protein and the dibenzocyclooctane derivative.

<Cell Crude cell extract and Cy5 .5- ADIBO Strain-facilitated Azid - Alkyne cyclization addition ( SPAAC )On by Conjugation  Results Analysis>

In this example, to test the specificity of SPAAC, the same conjugation reaction as the conjugation reaction performed on the purified GST-F47AZF was performed on cell extracts prepared from GST-F47AZF expressing cells. Cells expressing wild-type GST, cells expressing mutant GST (GST-F47AZF) in the presence of 1 mM AZF, and cells expressing GST-F47AZF were cultured in the absence of AZF, . 5-ADIBO was added. At this time, 15 μL of a 10 mM phosphate buffer (pH 7.0) mixture containing 10 μL of each cell-cell extract, 100 mM NaCl and 200 μM Cy5.5-ADIBO was prepared and used. Thereafter, the mixture was incubated at 37 ° C for 1 hour and then subjected to SDS-PAGE.

As shown in the fluorescence gel image (A) and the Coomassie stained gel image (B) of FIG. 5, in the lysates of the GST-F47AZF expressing cells, a pronounced conjugation reaction with the target protein was observed. However, Conjugation reaction was not observed in the GST-F47AZF expressing cells or the wild-type GST expressing cell lysates. In addition, minimal background fluorescence due to nonspecific conjugation reactions was observed, which means that SPAAC is specific to target proteins, including AZF.

< Conjugation  Sensitivity Analysis of Reaction>

In this example, the reactants between GST-F47AZF and Cy5.5-ADIBO according to sequential dilution were analyzed. Figure 6 shows the fluorescence scan gel image (top) and Coomassie stained gel image (bottom) of the GST-F47AZF and Cy5.5-ADIBO conjugation reagents. As shown in Fig. 6, a small amount of the label protein as low as 8 ng to 20 ng could be detected.

In order to demonstrate that the protein labeling method of the present invention can be used for the analysis of protein expression levels, expression of GST-F47AZF was carried out under different concentrations of L-arabinose. The lysates of GST-F47AZF expressing cells were treated with the labeling reagent Cy5.5-ADIBO and then analyzed by anti-body Western blotting by SDS-PAGE (Fig. 7).

As shown in Figure 7, there was a pronounced difference corresponding to the predicted expression level based on the given L-arabinose concentration. By comparison, the same expression patterns were observed in fluorescence and Coomassie stained gel images. These results indicate that the protein expression level analysis using the protein labeling method of the present invention is not as sensitive as the antibody-based Western blot (generally detectable up to 0.1 ng when using a chemiluminescent substrate), but is much more sensitive than Coomassie staining And the specificity was also excellent.

It will be understood by those of ordinary skill in the art that the foregoing description of the embodiments is for illustrative purposes and that those skilled in the art can easily modify the invention without departing from the spirit or essential characteristics thereof. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

The scope of the present invention is defined by the appended claims rather than the detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be interpreted as being included in the scope of the present invention .

Claims (20)

delete delete delete delete delete delete delete delete delete delete delete Replacing the amino acid contained in the target protein with an azide group-containing unnatural amino acid;
(2-azidethoxy) carbonyl] -l-lysine, which is an azido group-containing unnatural amino acid, contained in the labeling reagent containing a cycloalkynyl group and the above-mentioned cycloalkynyl group Labeling the target protein with the labeling reagent through a conjugation reaction between azide groups; And
Purifying the labeled target protein
/ RTI &gt; of the protein.
13. The method of claim 12,
Further comprising quantitatively or qualitatively analyzing the purified target protein.
A dye, and a labeling reagent formed by reacting a cycloalkynyl group-containing compound,
The azido group of the target protein comprising the cycloalkynyl group contained in the labeling reagent and the azide group-containing unnatural amino acid causes a conjugation reaction to thereby label the target protein, The target protein is used to purify the target protein and to analyze the target protein.
Composition for protein analysis.
15. The method of claim 14,
Wherein the cycloalkynyl group may contain a heteroatom.
15. The method of claim 14,
Wherein the cycloalkynyl group comprises a cyclooctynyl group.
15. The method of claim 14,
Wherein the cycloalkynyl group-containing compound comprises dibenzocyclooctane, or a dibenzocyclooctane derivative.
15. The method of claim 14,
Wherein the labeling reagent comprises a Cy5.5-linked aza-dibenzocyclooctane (Cy5.5-ADIBO).
15. The method of claim 14,
Wherein the conjugation reaction is carried out by a strain-promoted azide-alkyne cycloaddition (SPAAC) between the cycloalkynyl group and the azide group contained in the unnatural amino acid , Composition for protein analysis.
15. The method of claim 14,
Wherein the dye comprises a radioactive substance, a fluorescent dye, or a biological dye.

Images