KR20180070985A - Fret sensor for detecting l-glutamine and detecting method of l-glutamine using the same - Google Patents

Abstract

The present invention relates to a FRET sensor for detecting L-glutamine, an L-glutamine detection method using the FRET sensor for detecting L-glutamine, and a composition for detecting L-glutamine including the FRET sensor for detecting L-glutamine. The FRET sensor for detecting L-glutamine comprises: an L-glutamine-binding protein into which a fluorescent donor is introduced; and a fluorescent receptor protein.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a FRET sensor for detecting L-glutamine and a method for detecting L-glutamine using the same,

The present invention relates to a L-glutamine detecting FRET sensor, a L-glutamine detecting FRET sensor, an L-glutamine detecting method, and a L-glutamine detecting FRET sensor.

Fluorescence resonance energy transfer (FRET) has been widely used in fluorescence-based applications due to the peculiar dependence of their distance. FRET occurs between two fluorophores when there is a significant overlap between the emission spectrum of one fluorophore and the other absorption spectrum and when the distance between the fluorophores is generally less than 10 nm do. Given that FRET efficiency is inversely proportional to the sixth power of the separation distance, the FRET is very suitable for detecting molecular interactions and spatial changes. FRET-based sensors are becoming increasingly popular due to the development of fluorescent protein of various colors. Recombinant fluorescent protein FRET pairs may be expressed independently of each other, or may be in a fused form. These FRET pairs have been used to detect various biomolecules and ions and to measure the activity of enzymes. Although a variety of FRET sensors based on fluorescent proteins have been developed and successfully applied to a variety of applications, the FRET sensor method has inherent limitations, including the large size of fluorescent proteins and the need for N- or C-terminal fusion in general. Due to these constraints, many FRET sensors only show a degree of change in the FRET signal that is barely above the noise.

Over the past decade, several fluorescent amino acids have been genetically incorporated into cellular proteins using orthogonal aminoacyl-tRNA / aminoacyl-tRNA synthetase (aatRNA) / aminoacyl-tRNA synthetase, aaRS] Respectively. These amino acids have been used to investigate protein-folding conditions, interactions between proteins and ligands, cellular localization of various proteins, and protein fusion. Recently, one of the fluorescent amino acids was used for analyzing protein-nucleic acid interactions by FRET, and the dissociation constant was determined by using a method known as electrophoretic mobility shift assay, such as electrophoretic mobility shift assay .

The genetic introduction of fluorescent amino acids has two important advantages over FRET-based biochemical studies:

(1) the introduction of the fluorescent protein is limited to the N-terminal or C-terminal, while the amino acid can be incorporated at any position of the protein.

(2) The incorporation of these amino acids is achieved with amino acid mutations, thus causing minimal perturbation to the protein.

Korean Patent Publication No. 2010-0117877 discloses a method of detecting a ligand using a FRET biosensor.

The present invention provides a composition for detecting L-glutamine comprising a FRET sensor for detecting L-glutamine, an L-glutamine detecting method using the FRET sensor for detecting L-glutamine, and a FRET sensor for detecting L-glutamine.

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.

The first aspect of the present application relates to a L-glutamine-binding protein into which a fluorescent donor is introduced; And a FRET sensor for detecting L-glutamine, which comprises a fluorescent receptor protein.

A second aspect of the present invention provides a method for detecting L-glutamine using a FRET sensor for detecting glutamine according to the first aspect of the present invention.

A third aspect of the present invention provides a composition for detecting L-glutamine, comprising a FRET sensor for detecting glutamine according to the first aspect of the present invention.

According to one embodiment of the present invention, a FRET pair is formed by introducing a fluorescent unnatural amino acid into an L-glutamine-binding protein and fusing the L-glutamine-binding protein together with a green fluorescent protein to form L - A FRET sensor capable of selectively detecting glutamine can be produced.

The FRET sensor for detecting L-glutamine according to an embodiment of the present invention shows no change to the remaining 19 natural amino acids except for L-glutamine or D-glutamine, and selectively acts on L-glutamine.

The FRET sensor for detecting L-glutamine according to an embodiment of the present invention has an advantage that a fluorescent unnatural amino acid can be located at any position of the target protein and a single fluorescent protein is used as a FRET receptor. This is to improve the limited size of large size and fusion, which is a disadvantage of conventional FRET sensors requiring two fluorescent proteins.

According to one embodiment of the present application, the use of rapamycin complex 1 (mTORC1), which is used as a carbon source to supply building blocks for protein biosynthesis and substrates for the TCA cycle, and which is associated with human disease, including diabetes and cancer Glutamine < / RTI > associated with the regulation of L-glutamine, thereby enabling cell imaging in biomolecules or living cells.

1 is a schematic diagram of a FRET sensor for L-glutamine detection in one embodiment of the present invention.
Figure 2 is a graph showing the normalized absorption and emission spectra of CouA and EGFP in one embodiment of the invention.
3A is an image showing SDS-PAGE results of a FRET sensor for detecting L-glutamine in one embodiment of the present invention.
3B is a fluorescence spectrum of a FRET sensor EGFP-GlnBP-F136CouA for L-glutamine detection in one embodiment of the present invention.
3C is a fluorescence spectrum of a FRET sensor EGFP-GlnBP-N138CouA for L-glutamine detection in one embodiment of the present invention.
4A to 4F are graphs showing MALDI-TOF MS analysis results of trypsin-degraded EGFP-GlnBP-WT and EGFP-GlnBP mutant proteins containing CouA in one embodiment of the present invention to be.
5 (a) and 5 (b) are graphs showing SDS-PAGE analysis results of a FRET sensor for detecting L-glutamine in one embodiment of the present invention.
FIG. 6 is a graph showing changes in FRET ratio (I _EGFP / I _CouA ) of the FRET sensor for detecting L-glutamine according to L-Gln binding in one embodiment of the present _invention .
7A and 7B are graphs showing the dependency of the FRET ratio change of the FRET sensor for L-glutamine detection on the pH and salt concentration in one embodiment of the present invention.
8 (a) and 8 (b) are graphs showing changes in fluorescence spectrum (a) and FRET ratio (b) of L-glutamine detecting FRET sensor EGFP- _10- GlnBP-N138CouA under the optimum condition FIG.
FIG. 9 is a graph showing selectivity analysis of the natural amino acid and D-Gln of the FRET sensor for detecting L-glutamine EGFP- _10- GlnBP-N138CouA in one embodiment of the present invention.
10 (a) and 10 (b) are graphs showing changes in fluorescence spectrum (a) and FRET ratio (b) of L-glutamine detecting FRET sensor EGFP-10-GlnBP-N138CouA titrated with FBS FIG.

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 (s) thereof " included in the expression of the machine form means a mixture or combination of one or more elements selected from the group consisting of the constituents described in the expression of the form of a marker, Quot; means at least one selected from the group consisting of the above-mentioned elements.

Throughout this specification, the description of "A and / or B" means "A or B, or A and B".

Throughout this specification, " unnatural amino acid " refers to 20 common amino acids, non-pyrocidin or amino acids that are not selenocysteine; Other terms that can be used in a similar sense to the term " unnatural amino acid " include " non-cirrhically coded amino acid ", " naturally occurring amino acid ", and the like. 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 or pyriocin or selenocysteine.

Throughout this specification, "nucleic acid" refers to deoxyribonucleosides, deoxyribonucleotides, ribonucleosides, ribonucleotides, and polymers thereof in single- or double-stranded form. Unless specifically limited otherwise, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties to reference nucleic acids and are metabolized in a manner similar to that of naturally occurring nucleotides. Unless otherwise specifically limited, the term also refers to oligonucleotides, including PNA (peptidic nucleic acid), DNA analogs used in antisense technology (phosphorothioate, porphloamidate, etc.). Unless otherwise specified, a particular nucleic acid sequence also includes its conservatively modified variants and complementary sequences, as well as clearly specified sequences.

Throughout this specification, "polypeptide", "peptide", and "protein" are used interchangeably herein and refer to a polymer of amino acid residues. That is, the description of the protein equally applies to the description of the peptide and the description of the polypeptide, and vice versa. The term applies to naturally occurring amino acid polymers as well as to amino acid polymers wherein one or more amino acid residues are unnatural amino acids. As used herein, the term includes amino acid chains of any length, in which the amino acid residues are joined by a covalent peptide bond, including full length proteins.

Throughout the present specification, the description of FRET (fluorescence resonance energy transfer) is based on the assumption that, when a donor which is a short wavelength fluorescent protein absorbs energy from the outside, the excitation energy of the donor is released as light energy, Refers to a phenomenon that an acceptor, which is a long wavelength fluorescent protein positioned within a predetermined distance, is transmitted without luminescence and only long wavelength fluorescent light of the receptor is emitted. The donor and acceptor should be located at a very short distance (less than 10 nm) in order to generate receptor fluorescence by FRET or photobleaching of donor emitting light. When the receptor, which is a fluorescent protein capable of absorbing energy by causing resonance with the donor and a fluorescent protein that emits light of a specific wavelength, comes close to a predetermined distance, light energy irradiated to excite the donor from the outside Is transmitted from the donor to the receptor without luminescence to decrease the emission of the specific wavelength of the donor and FRET phenomenon occurs in which the receptor that receives energy from the donor emits light in its own wavelength band.

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

The first aspect of the present application relates to a L-glutamine-binding protein into which a fluorescent donor is introduced; And a FRET sensor for detecting L-glutamine, which comprises a fluorescent receptor protein.

In one embodiment of the present invention, the FRET sensor for detecting L-glutamine comprises the steps of: preparing an expression vector that expresses a fusion protein of the fluorescent receptor protein and the L-glutamine-binding protein; Replacing part of the nucleic acid sequence of the expression vector with a termination codon; Introducing the fluorescent donor into the expression vector; And co-transforming the expression vector with a host cell to obtain a FRET sensor.

In one embodiment of the present invention, the FRET sensor for detecting L-glutamine shows no change with respect to the remaining 19 natural amino acids except for L-glutamine or D-glutamine, and may selectively act on L-glutamine . For example, the FRET sensor for detecting L-glutamine is structurally changed by L-glutamine and at the same time FRET ratio is increased. Thus, L-aspartic acid (L-Asn), D- Only L-glutamine can be selectively detected with respect to other amino acids including glutamine.

In one embodiment herein, the nucleic acid sequence may be optionally or nonselectively modified in accordance with methods known in the art, for example from the group consisting of TAG, TGA, TAA, and combinations thereof May be substituted by the termination codon selected.

In one embodiment of the invention, the substitution of the nucleic acid sequence may be performed by a site-specific mutation.

In one embodiment of the invention, the host cell may be E. coli. For example, a plasmid containing a gene of a protein having a codon substituted with the termination codon, a plasmid containing a suitable tRNA gene, and a plasmid containing a suitable aminoacyl tRNA synthetase gene can be used as a host cell, To prepare a transformed E. coli, and culturing the transformed E. coli in a medium containing the fluorescent donor, but the present invention is not limited thereto.

In one embodiment herein, the fluorescent donor may be an unnatural amino acid, for example, the unnatural amino acid is L- (7-hydroxycoumarin-4-yl) ethylglycine [L- (7-hydroxycoumarin -4-yl) ethylglycine], 3- (6-acetylnaphthalen-2-ylamino) -2-aminopropanoic acid, yl alanine [acridon-2-ylalanine], and combinations thereof. The term " amino acid "

For example, the unnatural amino acid can bind to tRNA by the aminoacyl tRNA synthetase, and the tRNA bound to the unnatural amino acid recognizes the substituted nucleic acid sequence, But is not limited to, coding. 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 " means a method using a cellular translation system, including, but not limited to, using one or more of polynucleotides, codons, tRNAs, and ribosomes.

In a conventional FRET sensor, the introduction of a fluorescent protein is limited to the N-terminus or the C-terminus, whereas in one embodiment of the present invention, the fluorescent unnatural amino acid as the fluorescent donor is the L- Any place can be incorporated. For example, the fluorescent unnatural amino acid as the fluorescent donor can be introduced through gene fusion at the F136 or N138 position of the L-glutamine-binding protein.

In one embodiment of the present invention, the L-glutamine-binding protein comprises SEQ ID NO: 2 and the fluorescent donor is introduced at the F136 or N138 position in the L-glutamine-binding protein of SEQ ID NO: .

In one embodiment of the invention, the introduction of the fluorescent donor may be accomplished by amino acid mutagenesis.

In one embodiment, the fluorescent receptor protein is selected from the group consisting of a green phosphor protein (GFP), a modified green phosphor protein (mGFP), an enhanced green phosphor protein (eGFP), a red phosphor protein (RFP) (mRFP), Enhanced Red Phosphor Protein (eRFP), Blue Phosphor Protein (BFP), Modified Blue Phosphor Protein (mBFP), Enhanced Blue Phosphor Protein (eBFP), Yellow Phosphor Protein (mYFP), enhanced yellow fluorescent protein (eYFP), blue fluorescent protein (CFP), modified blue fluorescent protein (mCFP), enhanced blue fluorescent protein (eCFP), and combinations thereof May include.

In one embodiment of the invention, the fluorescent receptor protein may be linked to the N-terminus of the L-glutamine-binding protein. For example, the fluorescent receptor protein may specifically be an enhanced green fluorescent protein, and the enhanced green fluorescent protein is fused to the N-terminus of the L-glutamine-binding protein to form a FRET pair Lt; / RTI >

In one embodiment of the present invention, the FRET sensor for L-glutamine detection may be affected by pH and / or salt concentration, but may not be limited thereto. For example, the FRET sensor for L-glutamine detection may depend on the pH and / or the salt concentration, and the FRET ratio of the FRET sensor for detecting L-glutamine may vary. For example, the FRET ratio increases as the pH increases to about 9, and when the pH is above about 9, the change in FRET ratio decreases with deprotonation of the a-amino group of L-glutamine But may not be limited thereto.

The second aspect of the present invention provides a method for detecting L-glutamine using the FRET sensor for detecting L-glutamine according to the first aspect of the present invention. The detailed description of the method for detecting L-glutamine according to the second aspect of the present application is omitted for the sake of simplicity of description of the first aspect of the present application. However, The same can be applied to the second aspect.

In one embodiment of the present invention, the FRET sensor for detecting L-glutamine comprises an L-glutamine-binding protein into which a fluorescent donor is introduced; And a fluorescent receptor protein.

In one embodiment of the present invention, the method for detecting L-glutamine using the FRET sensor for detecting L-glutamine comprises the steps of preparing an expression vector expressing the fusion protein of the fluorescent receptor protein and the L-glutamine-binding protein ; Replacing part of the nucleic acid sequence of the expression vector with a termination codon; Introducing the fluorescent donor into the expression vector; Co-transforming said expression vector with a host cell to obtain a FRET sensor; And detecting L-glutamine through the reaction of the obtained FRET sensor and L-glutamine.

In one embodiment of the present invention, the detection of L-glutamine may be performed by measuring a change in the FRET ratio of the FRET sensor according to the concentration of L-glutamine. For example, the change of the FRET ratio is determined by measuring the FRET ratio (R _x ) when the concentration of L-glutamine is x with respect to the FRET ratio (R ₀ ) when the concentration of L-glutamine is 0 But may be, but not limited to:

In one embodiment of the present invention, the FRET sensor for detecting L-glutamine shows no change with respect to the remaining 19 natural amino acids except for L-glutamine or D-glutamine, and may selectively act on L-glutamine . For example, the FRET sensor for detecting L-glutamine is structurally changed by L-glutamine and at the same time FRET ratio is increased. Thus, L-aspartic acid (L-Asn), D- Only L-glutamine can be selectively detected with respect to other amino acids including glutamine.

In one embodiment herein, the nucleic acid sequence may be optionally or nonselectively modified in accordance with methods known in the art, for example from the group consisting of TAG, TGA, TAA, and combinations thereof May be substituted by the termination codon selected.

In one embodiment of the invention, the substitution of the nucleic acid sequence may be performed by a site-specific mutation.

In one embodiment of the invention, the host cell may be E. coli. For example, a plasmid containing a gene of a protein having a codon substituted with the termination codon, a plasmid containing a suitable tRNA gene, and a plasmid containing a suitable aminoacyl tRNA synthetase gene can be used as a host cell, To prepare a transformed E. coli, and culturing the transformed E. coli in a medium containing the fluorescent donor, but the present invention is not limited thereto.

In one embodiment herein, the fluorescent donor may be an unnatural amino acid, for example, the unnatural amino acid is L- (7-hydroxycoumarin-4-yl) ethylglycine [L- (7-hydroxycoumarin -4-yl) ethylglycine], 3- (6-acetylnaphthalen-2-ylamino) -2-aminopropanoic acid, yl alanine [acridon-2-ylalanine], and combinations thereof. The term " amino acid "

For example, the unnatural amino acid can bind to tRNA by the aminoacyl tRNA synthetase, and the tRNA bound to the unnatural amino acid recognizes the substituted nucleic acid sequence, But is not limited to, coding. 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 " means a method using a cellular translation system, including, but not limited to, using one or more of polynucleotides, codons, tRNAs, and ribosomes.

In a conventional FRET sensor, the introduction of a fluorescent protein is limited to the N-terminus or the C-terminus, whereas in one embodiment of the present invention, the fluorescent unnatural amino acid as the fluorescent donor is the L- Any place can be incorporated. For example, the fluorescent unnatural amino acid as the fluorescent donor can be introduced through gene fusion at the F136 or N138 position of the L-glutamine-binding protein.

In one embodiment of the present invention, the L-glutamine-binding protein comprises SEQ ID NO: 2 and the fluorescent donor is introduced at the F136 or N138 position in the L-glutamine-binding protein of SEQ ID NO: .

In one embodiment of the invention, the introduction of the fluorescent donor may be accomplished by amino acid mutagenesis.

In one embodiment, the fluorescent receptor protein is selected from the group consisting of a green phosphor protein (GFP), a modified green phosphor protein (mGFP), an enhanced green phosphor protein (eGFP), a red phosphor protein (RFP) (mRFP), Enhanced Red Phosphor Protein (eRFP), Blue Phosphor Protein (BFP), Modified Blue Phosphor Protein (mBFP), Enhanced Blue Phosphor Protein (eBFP), Yellow Phosphor Protein (mYFP), enhanced yellow fluorescent protein (eYFP), blue fluorescent protein (CFP), modified blue fluorescent protein (mCFP), enhanced blue fluorescent protein (eCFP), and combinations thereof May include.

In one embodiment of the invention, the fluorescent receptor protein may be linked to the N-terminus of the L-glutamine-binding protein. For example, the fluorescent receptor protein may specifically be an enhanced green fluorescent protein, and the enhanced green fluorescent protein is fused to the N-terminus of the L-glutamine-binding protein to form a FRET pair Lt; / RTI >

In one embodiment of the present invention, the detection of L-glutamine may be affected by, but not limited to, pH and / or salt concentration. For example, the FRET sensor for L-glutamine detection may be dependent on the pH and / or salt concentration, and the FRET ratio of the FRET sensor for detecting L-glutamine may be changed. For example, 9, the FRET ratio is increased. When the pH is more than about 9, the change in the FRET ratio may be reduced by deprotonation of the? -Amino group of L-glutamine, .

A third aspect of the present invention provides a composition for detecting L-glutamine, comprising a FRET sensor for detecting glutamine according to the first aspect of the present invention.

The detailed description of the composition for the detection of L-glutamine according to the third aspect of the present application will be omitted from the description of the first aspect of the present application. However, The same applies to the third aspect of FIG.

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are given to aid understanding of the present invention, and the present invention is not limited to the following examples.

[Example]

All chemicals and DNA oligomers commercially available were used without further purification. MALDI-TOF MS was obtained using a Bruker Autoflex Speed Series mass spectrometer (Bruker Daltonics, Leipzig, Germany).

All UV-visible absorption spectra were measured with a Jasco V-660 spectrophotometer, and fluorescence spectra were taken with a Hitachi F-7000 fluorescence spectrophotometer.

Expression and purification of FRET sensor

The GlnBP gene was amplified from E. coli genomic DNA, and the EGFP gene was obtained commercially by gene synthesis. EGFP-GlnBP fusion genes with different linker lengths were synthesized by overlap extension PCR and inserted into pBAD / Myc-His (Invitrogen) to generate pBAD-EGFP-GlnBP-WT. The amber codon (TAG) was introduced at position 136 or 138 of GlnBP by a site-specific mutation. In order to express EGFP-GlnBP fusion protein containing CouA, pBAD-EGFP-GlnBP with an amber codon was cotransformed with E. coli C321.A strain (Addgene) together with pEvol-CouRS26. Cells were amplified in LB medium (lysogeny broth) supplemented with ampicillin (100 μg / mL) and chloramphenicol (35 μg / mL). Seed culture (2 mL) was amplified with either ampicillin (100 μg / mL), chloramphenicol mL), and 100 mL LB medium supplemented with 1 mM CouA. Protein expression was induced by adding 0.2% L-arabinose when the optical density reached 0.8, and the cultures were grown overnight at 37 ° C. Cells were harvested by centrifugation, and were re-suspended in lysis buffer _{(50 mM NaH 2 PO 4,} 300 mM NaCl, 10 mM imidazole, and pH 8.0), it was sonicated. The target protein was purified by Ni-NTA affinity chromatography under natural conditions according to the manufacturer's protocol (Qiagen). Purified FRET sensors was dialyzed against phosphate buffer _{(50 mM NaH 2 PO 4,} pH 8.0) for further experiments.

FRET sensor design

The FRET sensor used here was designed by introducing emerald GFP (emerald GFP, EGFP) into the L-glutamine-binding protein by gene incorporation and introducing the fluorescent amino acid into the gene fusion (Fig. 1). FIG. 1 is a schematic view of the designed FRET sensor, wherein EGFP is fused to the N-terminal of GlnBP and CouA is incorporated at a position where the largest difference between CouA and EGFP occurs due to ligand binding. Among the above-mentioned fluorescent amino acids that have been successfully incorporated into the protein by the gene introduction, L- (7-hydroxycoumarin-4-yl) ethylglycine [L- (7-hydroxycoumarin- Were chosen for the present application (Fig. 2), since the emission spectrum of EGFP exhibited significant overlap with the absorption spectrum of EGFP. Figure 2 shows the canonical absorption and emission spectra for CouA and EGFP.

In addition, the aatRNA / aaRS pair for CouA is consistent with protein expression in bacterial cells, and the amino acid could be easily prepared in both synthetic steps. As a sensor protein, the L-glutamine binding protein (GlnBP) from E. coli was selected because these proteins are structurally well characterized and have been used as fluorescence sensors for L-Gln. GlnBP is a small periplasmic binding protein with 226 amino acid residues and has a dissociation constant of sub-micromolar at a 1: 1 molar ratio and binds to L-Gln. L-Gln is used as a building block for protein biosynthesis and as a carbon source to supply substrates for the TCA cycle. In addition, the L-Gln is involved in the modulation of rapamycin complex 1 (mTORC1), a regulatory disorder associated with human disease, including diabetes and cancer. Therefore, the detection of L-Gln is important in studying the biochemical function of L-Gln.

The crystal structure of GlnBP is reported in two forms, ligand-free and ligand-binding, indicating that the L-Gln causes significant structural changes with ligand binding. Based on the structure of L-Gln as described above, the optimal position for incorporation of GFP fusion and CouA was investigated in order to maximize the FRET change according to the binding of the ligand. GlnBP contains two domains linked by two hinges. The positions of the hinge are Y86, K87 and Q183 and Q184. The N- and C-termini are on the same domain, suggesting that the unique fluorescent protein is a FRET pair and that fusion to each terminus is unlikely to cause significant FRET changes (Fig. 1). The N-terminal is located at one edge of the entire protein structure, and the C-terminal is located near the hinge region. Because the C-terminus is located at the border of the two domains, such a C-terminus position will be inadequate for GFP fusion as it is not sensitive to structural changes. Thus, the GFP fused at the N-terminus to maximize the FRET change.

Next, the location for CouA incorporation was explored by comparing the two structural forms of GlnBP. The superimposition of the structure revealed that the structural change caused by ligand binding is due to the movement of V104 to L155 residues. To identify such residues that exhibit the greatest displacement from the N-terminus according to ligand binding, the alpha-carbon of Leu5, which is the first N-terminal residue visible in the structure, and the apo-form, The distance between the alpha-carbon of 104 to 155 residues of the two ligand-bound forms was measured. The distance difference between the two forms was calculated (Table 1). Table 1 below shows the measurement of the distance of V104 and L155 from Leu5 in GlnBP and the% change calculation at the above distance due to ligand binding. The distance between the α- carbon was measured using the PyMOL, 5 residues (S116, G117, F136, P137 , and N138) represents the largest change in distance was indicated in bold. The PDB ID used in the measurements is 1GGG (apo-form) and 1WDN (ligand-conjugated form).

Five residues (S116, G117, F136, P137, and N138) representing the most prominent distance change were considered as potential couA incorporation sites. Among these residues, S116 and G117 were located at the interface of the two domains near the ligand position in the ligand-bound form, which could potentially affect the binding affinity of the protein when they were replaced by CouA ; Therefore, they were not even considered. The remaining three residues were located in a loop between? -Strand and? -Helix. While Pro137 was excluded because it was expected to be structurally important in the loop, F136 and N138 were selected as sites for CouA incorporation.

CouA  Expression and purification of the included FRET sensor

To prepare the designed FRET sensor protein, a gene encoding C-terminal His ₆ -tagged GlnBP and a gene encoding EGFP were ligated so that the obtained protein could be C-terminally fused to EGFP. In addition, the codon for either F136 or N138 in GlnBP was replaced by the amber termination codon (TAG). CouA is N-Cbz-L- glutamic acid benzyl ester The method of reporting from the (N -Cbz-L-glutamic acid benzyl ester) [J. Wang, J. Xie and PG Schultz, J. Am. Chem . Soc . , 2006, 128 , 8738-8739. The EGFP-GlnBP construct containing CouA was expressed in the presence of CouA and the aaRNA / aaRS (CouRS) pair developed above. For this expression, E. coli strain C321.A derived from E. coli MG1655 was used. The mutant proteins EGFP-GlnBP-F136CouA and EGFP-GlnBP-N138CouA were expressed in the presence of the corresponding developed tRNA / aaRS pair and 1 mM CouA and purified by Ni-NTA affinity chromatography, The yields were 8 to 10 mg / L, respectively. For comparison, the yield of the EGFP-GlnBP fusion protein with the wild-type GlnBP sequence (except CouA) was 15-20 mg / L. As shown in FIG. 3A, analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed a band with a size corresponding to the full-length protein. The introduction of the fluorescent amino acid was confirmed by fluorescence images visualized by Coomassie-staining (left side in Fig. 3A) and fluorescence (right side in Fig. 3A). EGFP GlnBP WT was also analyzed as a control.

MALDI-TOF MS analysis of refined FRET sensor

After trypsin digestion, the incorporation of CouA was confirmed by MALDI-TOF MS (matrix-assisted laser desorption / ionization time-of-flight mass spectrometric) analysis. The FRET sensor (200 μg, final concentration 0.5 mg / mL) was digested with trypsin (20 μM) in a reaction buffer containing 50 mM Tris and 0.1% SDS for 12 hours at 37 ° C, 18 < / RTI > spin column. The desaturated tryptic digests were incubated with α-cyano-4-hydroxycinnamic acid (CHCA) matrix (50% acetonitrile and 1 (v / v) 10 mg / mL in water containing 10% trifluoroacetic acid) and tested for MALDI-TOF MS analysis. Peptide X (QFPNIDNAYMELGTNR) includes F136 and N138; Each residue was replaced by CouA in Peptide X-136CouA or X-138CouA. (A), EGFP-GlnBP-N138CouA (c), EGFP-GlnBP-N138CouA (d), EGFP-GlnBP- -7GlnBP-N138CouA (e), and EGFP-10-GlnBP-N138CouA (f), and the analysis confirmed the incorporation of CouA and found that there was no amino acid introduced at this position .

L-Gln titration and amino acid selectivity test

FRET sensors protein samples (100 nM) is the corresponding phosphate buffer _{(50 mM NaH 2 PO 4,} 0-500 mM NaCl, pH 7.5-10.5) L-Gln (0, 50, 100, 200, 500, 1000, 5000 in , 10000, 50000 and 100000 nM). Fluorescence was scanned at 420 nm to 580 nm at each concentration with excitation at 360 nm. The FRET ratio (R, I _{468 nm} / I _{518 nm} ) was calculated from the respective fluorescence spectra and the FRET ratio change was calculated by the following equation 1:

Where R ₀ is the FRET ratio at [L-Gln] = 0 and R _x is the FRET ratio at [L-Gln] = x. In the optimization experiments, fluorescence was measured with excitation at 360 nm at a fixed concentration of L-Gln (0 or 50 μM) at different pH or salt concentrations and the change in FRET ratio was calculated. For the amino acid selectivity test, the FRET ratio change to EGFP- _10- GlnBP-N138CouA (100 nM) as a FRET sen in the presence of 50 mM NaH ₂ PO ₄ (pH 9.0) containing 50 mM NaCl mu] M) and excitation at 360 nm. All measurements were performed independently 3 times, and the data were averaged. The dissociation constant of the FRET sensor was calculated using DynaFit.

Evaluation of FRET sensor

The FRET sensor containing CouA was investigated for their FRET changes with ligand binding. 3b and 3c show fluorescence spectra for EGFP-GlnBP-F136CouA (b) and EGFP-GlnBP-N138CouA (c), respectively. The protein (100 nM) was titrated with L- (Assay conditions: 100 nM EGFP-GlnBP mutant protein, 50 mM phosphate buffer (pH 8.0), 300 mM NaCl, and L-Gln (indicated concentrations)] at 400 nm to 580 nm, . As shown in Figures 3b and 3c, EGFP release by FRET was observed for both mutants. However, with the increasing concentration of L-Gln, only the above FRET ratio of EGFP-GlnBP-N138CouA (I _{EGFP, 518 nm} / I _{CouA , 468 nm} ) increased. The ratio increased 1.4-fold when the protein was saturated with L-Gln. In the case of the F136 mutation, the fluorescence maxima of CouA were shifted from 450 nm to 420 nm, which reduced spectral overlap with the absorption spectrum of EGFP and resulted in a reduced FRET signal. The fluorescence spectrum of 8-hydroxycoumarin has been reported to be blue-citrated when the fluorophore interacts with polar residues (hydrogen bond donor or acceptor) in the protein. Thus, CouA in EGFP-GlnBP-F136CouA forms a complex with residues near F136, and the inventors expect that the complex will be stabilized by binding of L-Gln. This hybridization shifted the fluorescence spectrum of CouA to shorter wavelengths and reduced the spectral overlap of EGFP with the absorption spectrum.

Redesign of FRET sensor

Although the EGFP-GlnBP-N138CouA shows a significant increase in the FRET ratio with ligand binding, the sensor was expected to be designed to further increase the FRET rate change. Based on the crystal structure of GlnBP, N138 was expected to be the optimal moiety for replacement with CouA, as described above. Thus, the authors of the present invention engineered the amino acid sequence between EGFP and GlnBP. In the crystal structure of the GFP, some C-terminal residues are absent, presumably because the C-terminal residues are flexible, which precludes their modeling. Elimination of such residues would solidify the linker region and / or reduce the distance between CouA and EGFP, which was expected to increase the FRET rate change. The FRET sensor protein has been redesigned based on this structural consideration; The four mutant genes were constructed as shown in Table 2 below to have different linker lengths and the codon for N138 was replaced by an Ammerm codon to enable incorporation of CouA.

Figure 5 shows the results of an SDS-PAGE analysis of purified FRET sensor proteins expressed as described above, wherein EGFP-GlnBP mutant proteins with different lengths of linker were incubated with 1 mM CouA and the required developed tRNA / aaRS pair , And then purified by Ni-NTA affinity chromatography. The purified FRET sensor protein was analyzed by SDS-PAGE and visualized by Coomassie-staining (Fig. 5 (a)) and fluorescence (Fig. 5 (b)). As shown in Figure 5, their yield and purity were consistent with the yield and purity of FRET sensor proteins initially designed.

After trypsin digestion, incorporation of CouA was evidenced by MALDI-TOF MS (FIG. 4). The redesigned FRET sensor protein was evaluated by measuring fluorescence at different concentrations of L-Gln. From this fluorescence spectrum, the change in FRET ratio (I _EGFP / I _CouA ) (ΔR / R ₀ ) was calculated and plotted against L-Gln concentration (FIG. I _CouA and I _EGFP were measured at 468 nm and 518 nm after excitation at 360 nm and each data point represents the average of three independent experiments [Analysis conditions: 100 nM EGFP-GlnBP mutant protein, 50 mM phosphate buffer (pH 8.0), 300 mM NaCl, and L-Gln (indicated concentrations). The FRET ratio increased with increasing concentrations of L-Gln for all redesigned FRET sensor proteins, and the increase was increased as the linker length decreased. The best FRET sensor protein was EGFP- _10- GlnBP-N138CouA; Its FRET ratio increased 1.8-fold with L-Gln binding. The authors of the present application did not make the linker length shorter because the A226, A227, and G228 residues in EGFP were within the defined structure, and removal of such residues would affect the structure and function of the FRET sensor protein It was expected.

Dependence of FRET sensor on salt concentration and pH

In order to test whether the FRET sensor has dependency on salt concentration or pH, the presence of L-Gln (50 μM) and the pH in the absence (FIG. 7 (a)) and the salt concentration )] Value, and the change in FRET ratio obtained was calculated. FRET ratios were calculated from I _{CouA , 468 nm} and I _{EGFP , 518 nm in} the presence (50 μM) or absence of L-Gln according to excitation at 360 nm. 7 (a) conditions were 100 nM EGFP- ₁₀ -GlnBP-N138CouA, 50 mM phosphate buffer (at the indicated pH), 300 mM NaCl and conditions of Figure 7 (b) were 100 nM EGFP- _10- GlnBP -N138CouA, 50 mM phosphate buffer (pH 9.0), NaCl at the indicated concentration. Each data-point averaged three independent experiments.

Although a slight increase was observed with increasing concentrations from 0 mM to 50 mM, the sodium chloride concentration in the range of 0 to 500 mM did not significantly affect the FRET ratio change. However, the pH dependency was significant: as the pH was increased to pH 9, the FRET ratio change was increased. This is because in the 7-hydroxy coumarin the hydroxy group has a pk _a of about 8 and deprotonation of the hydroxy group increases fluorescence intensity at 460 nm. The decrease in the FRET rate change at higher pH (above 9.5) was due to the deprotonation of the _a -amino group of L-Gln (pKa = 9.1) and the reduction obtained in their binding affinity for the sensor protein will be.

Evaluation of FRET sensors under optimal conditions

Under optimal conditions (pH 9 and 50 mM sodium chloride), EGFP- _10- GlnBP-N138CouA was titrated to L-Gln and its fluorescence spectrum was obtained at increasing concentrations of L-Gln after excitation at 360 nm (Fig. 8 (a)).

Figure 8 (b) shows the change in FRET ratio _{, which} was calculated from I _{CouA , 468 nm} and I _{EGFP , 518 nm} after excitation at 360 nm. Analysis conditions: 100 nM EGFP- _10- GlnBP-N138CouA, 50 mM phosphate buffer (pH 9.0), 50 mM NaCl, and L-Gln at the indicated concentrations. Each data-point represents the average of three independent experiments. As shown in Figure 8 (b), the maximum FRET ratio change was 0.9, and the dissociation constant of L-Gln from the sensor protein was 396 +/- 77 nM, which was similar to the reported value (300 nM).

The selectivity of the sensor protein to other amino acids was then assessed. For all 20 amino acids, and D-Gln, the FRET ratio was determined by adding 50 [mu] M of each amino acid. Under the FRET ratio after excitation of 360 nm in the presence of L-Gln (50 μM) or _absence, I _{CouA, 468 nm,} and I _EGFP, was calculated from the _{518 nm} [Analysis _{conditions: 100 nM EGFP -10 -GlnBP-N138CouA} , 50 mM phosphate buffer (pH 9.0), 50 mM NaCl, and 50 [mu] M of each amino acid. Each data-point represents the average of three independent experiments. As shown in Fig. 9, no or only minimal change in the FRET ratio was observed for all amino acids except L-Gln. These results indicate that the designed sensor, EGFP- _10- GlnBP-N138CouA, responds to structural changes caused by L-Gln with an increase in FRET ratio of 1.9-fold and is similar to that of structurally similar amino acids (L-Asn, L- D-Gln). ≪ / RTI >

Next, the present inventors applied the sensor protein (EGFP- _10- GlnBP-N138CouA) to measure L-Gln concentration in a biological sample. 10 (a) and 10 (b) show fluorescence spectra and FRET ratio changes of EGFP- _10- GlnBP-N138CouA, respectively. Fluorescence spectra were measured at increasing concentrations of FBS after excitation at 360 nm and FRET ratio changes were calculated from I _{CouA , 468 nm} and I _{EGFP , 518 nm} after excitation at 360 nm. FBS concentration was normalized to logarithmic scale (assay conditions: 100 nM EGFP- _10- GlnBP-N138CouA, 50 mM phosphate buffer, pH 9.0, 50 mM NaCl, and FBS . Each data-point represents the average of three independent experiments. Fetal serum (FBS) is a widely used supplement for eukaryotic cell culture. L-Gln is the most abundant amino acid in serum and the concentration is known to be in the range of 500 μM to 1200 μM. From this titration experiment of the FRET sensor protein, the fluorescence spectrum was similar to that titrated with L-Gln. From these measurements, the concentration of L-Gln in FBS was estimated, which was approximately 500 [mu] M. These results indicate that the designed FRET sensor protein can be used to measure L-Gln concentration in biological sample complexes.

The distance between the fluorescent ends

The separation distance between CouA and the fluorescent end of EGFP was calculated for EGFP- _10- GlnBP-N138CouA. Based on the emission spectrum of CouA and the absorption spectrum of EGFP, the fluorescence distance was calculated (R ₀ = 49.2 Å). The FRET efficiency of the FRET sensor protein in the presence and absence of L-Gln was also calculated and was 0.284 and 0.153, respectively. From these values, the separation distance between the fluorophores was calculated; 65.5 A for the apo-form and 57.4 A for the ligand-binding A, which is a 12.4% change compared to the separation in the apo-form. It is known that the FRET change is maximized when the distance between two fluorophores approaches R ₀ . Therefore, when designing a FRET sensor, the change in FRET ratio can be maximized by placing CouA so that the change in distance before and after binding to the target material occurs near R ₀ .

As described above, FRET sensors based on the two fluorescent proteins used in the past have been useful tools for examining protein-protein interactions and structural changes in proteins, but the FRET sensors are inherently disadvantages Their large size and limited location for fusion (N- or C-terminal).

The FRET-based sensor of the present invention was developed by incorporating fluorescent unnatural amino acids into GlnBP as a FRET partner for GFP. GFP was fused to the N-terminus of GlnBP and the fluorescent unnatural amino acid CouA was introduced at the position for N138 in GlnBP. Optimal locations for CouA adoption were identified using structural information, distance calculation, and experimental evaluation. The FRET sensor protein was further optimized by changing the pH, salt concentration, and length of the linker between GFP and GlnBP. Under optimal conditions, the best FRET sensor did not show any slight change or change to the other 19 naturally occurring amino acids or D-Gln, but showed an increase of 1.9-fold in the FRET ratio with L-Gln binding. The novel FRET sensor according to the present invention can overcome the limitations of current FRET sensors that rely on two fluorescent proteins. Although one fluorescent protein in the novel FRET sensor is still required as a FRET receptor and it should also be located at one of the N- or C-terminus of the target protein, the fluorescent amino acid used as the FRET donor is not capable of undergoing the FRET change Lt; RTI ID = 0.0 > protein. ≪ / RTI > The incorporation of CouA is technically simple, and several locations can be simply tested to identify the optimal location. In addition, the structural information can be guided over the selection of location, significantly reducing the number of potential locations for experimental evaluation. Therefore, this method will be helpful for FRET sensor design, and the application of this method to cell imaging can be extended to methods for investigating biomolecular interactions in important biomolecules and living cells.

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 construed as being included within the scope of the present invention.

<110> Industry-University Cooperation Foundation Sogang University <120> FRET SENSOR FOR DETECTING L-GLUTAMINE AND DETECTING METHOD OF          L-GLUTAMINE USING THE SAME <130> DP20160518KR <160> 12 <170> KoPatentin 3.0 <210> 1 <211> 678 <212> DNA <213> Artificial Sequence <220> <223> Artificial GlnBP DNA seqeunce <400> 1 gcggataaaa aattagttgt cgcgacggat accgccttcg ttccgtttga atttaaacag 60 ggcgataaat atgtgggctt tgacgttgat ctgtgggctg ccatcgctaa agagctgaag 120 ctggattacg aactgaagcc gatggatttc agtgggatca ttccggcact gcaaaccaaa 180 aacgtcgatc tggcgctggc gggcattacc atcaccgacg agcgtaaaaa agcgatcgat 240 ttctctgacg gctactacaa aagcggcctg ttagtgatgg tgaaagctaa caataacgat 300 gtgaaaagcg tgaaagatct cgacgggaaa gtggttgctg tgaagagcgg tactggctcc 360 gttgattacg cgaaagcaaa catcaaaact aaagatctgc gtcagttccc gaacatcgat 420 aacgcctata tggaactggg caccaaccgc gcagacgccg ttctgcacga tacgccaaac 480 attctgtact tcatcaaaac cgccggtaac ggtcagttca aagcggtagg tgactctctg 540 gaagcgcagc aatacggtat tgcgttcccg aaaggtagcg acgagctgcg tgacaaagtc 600 aacggcgcgt tgaaaaccct gcgcgagaac ggaacttaca acgaaatcta caaaaaatgg 660 ttcggtactg aaccgaaa 678 <210> 2 <211> 226 <212> PRT <213> Artificial Sequence <220> <223> Artificial GlnBP Protein seqeunce <400> 2 Ala Asp Lys Lys Leu Val Val Ala Thr Asp Thr Ala Phe Val Phe   1 5 10 15 Glu Phe Lys Gln Gly Asp Lys Tyr Val Gly Phe Asp Val Asp Leu Trp              20 25 30 Ala Ala Ile Ala Lys Glu Leu Lys Leu Asp Tyr Glu Leu Lys Pro Met          35 40 45 Asp Phe Ser Gly Ile Pro Ala Leu Gln Thr Lys Asn Val Asp Leu      50 55 60 Ala Leu Ala Gly Ile Thr Ile Thr Asp Glu Arg Lys Lys Ala Ile Asp  65 70 75 80 Phe Ser Asp Gly Tyr Tyr Lys Ser Gly Leu Leu Val Met Val Lys Ala                  85 90 95 Asn Asn Asn Val Lys Ser Val Lys Asp Leu Asp Gly Lys Val Val             100 105 110 Ala Val Lys Ser Gly Thr Gly Ser Val Asp Tyr Ala Lys Ala Asn Ile         115 120 125 Lys Thr Lys Asp Leu Arg Gln Phe Pro Asn Ile Asp Asn Ala Tyr Met     130 135 140 Glu Leu Gly Thr Asn Arg Asa Asp Ala Val Leu His Asp Thr Pro Asn 145 150 155 160 Ile Leu Tyr Phe Ile Lys Thr Ala Gly Asn Gly Gln Phe Lys Ala Val                 165 170 175 Gly Asp Ser Leu Glu Ala Gln Gln Tyr Gly Ile Ala Phe Pro Lys Gly             180 185 190 Ser Asp Glu Leu Arg Asp Lys Val Asn Gly Ala Leu Lys Thr Leu Arg         195 200 205 Glu Asn Gly Thr Tyr Asn Glu Ile Tyr Lys Lys Trp Phe Gly Thr Glu     210 215 220 Pro Lys 225 <210> 3 <211> 714 <212> DNA <213> Artificial Sequence <220> <223> Artificial EGFP DNA seqeunce <400> 3 atgagtaaag gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt 60 gatgttaatg ggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc aacatacgga 120 aaacttaccc ttaaatttat ttgcactact ggaaaactac ctgttccatg gccaacactt 180 gtcactactt tcacctatgg tgttcaatgc tttgcgcgtt atccggatca catgaaacgg 240 catgactttt tcaagagtgc catgcccgaa ggttatgtac aggaacgcac tatatctttc 300 aaagatgacg ggaactacaa gacgcgtgct gaagtcaagt ttgaaggtga tacccttgtt 360 aatcgtatcg agttaaaagg tattgatttt aaagaagatg gaaacattct cggccataag 420 ctggaatata actacaacag ccacaaggtg tatatcaccg cggataagca gaagaatggc 480 atcaaggcca acttcaaaac ccgccacaac attgaagatg gatccgttca actagcagac 540 cattatcaac aaaatactcc aattggcgat ggccctgtcc ttttaccaga caaccattac 600 ctgtcgacac aatctgccct ttcgaaagat cccaacgaaa agcgtgacca catggtcctt 660 cttgagtttg taactgctgc tgggattaca catggcatgg atgagctcta caaa 714 <210> 4 <211> 238 <212> PRT <213> Artificial Sequence <220> <223> Artificial EGFP Protein seqeunce <400> 4 Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val   1 5 10 15 Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu              20 25 30 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys          35 40 45 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe      50 55 60 Thr Tyr Gly Val Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys Arg  65 70 75 80 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg                  85 90 95 Thr Ile Ser Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val             100 105 110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile         115 120 125 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn     130 135 140 Tyr Asn Ser His Lys Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly 145 150 155 160 Ile Lys Ala Asn Phe Lys Thr Arg His Asn Ile Glu Asp Gly Ser Val                 165 170 175 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro             180 185 190 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser         195 200 205 Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val     210 215 220 Thr Ala Gly Ily Thr Gly Met Asp Glu Leu Tyr Lys 225 230 235 <210> 5 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Artificial EGFP-GlnBP overlap Forward primer seqeunce <400> 5 ggcatggatg agctctacaa aggtaaatta gttgtcgcga cgg 43 <210> 6 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Artificial EGFP-GlnBP overlap Reverse primer seqeunce <400> 6 ccgtcgcgac aactaattta cctttgtaga gctcatccat gcc 43 <210> 7 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Artificial EGFP-4-GlnBP overlap Forward primer seqeunce <400> 7 gctgggatta cacatggcat ggatggtaaa ttagttgtcg cgacgg 46 <210> 8 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Artificial EGFP-4-GlnBP overlap Reverse primer seqeunce <400> 8 ccgtcgcgac aactaattta ccatccatgc catgtgtaat cccagc 46 <210> 9 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Artificial EGFP-7-GlnBP overlap Forward primer seqeunce <400> 9 gtaactgctg ctgggattac acatggtaaa ttagttgtcg cgacgg 46 <210> 10 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Artificial EGFP-7-GlnBP overlap Reverse primer seqeunce <400> 10 ccgtcgcgac aactaattta ccatgtgtaa tcccagcagc agttac 46 <210> 11 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Artificial EGFP-10-GlnBP overlap Forward primer seqeunce <400> 11 ccttcttgag tttgtaactg ctgctggggg taaattagtt gtcgcgacgg 50 <210> 12 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Artificial EGFP-10-GlnBP overlap Reverse primer seqeunce <400> 12 ccgtcgcgac aactaattta cccccagcag cagttacaaa ctcaagaagg 50

Claims (9)

An L-glutamine-binding protein into which a fluorescent donor is introduced; And a FRET sensor for detecting L-glutamine.
The method according to claim 1,
Wherein said fluorescent donor is an unnatural amino acid.
3. The method of claim 2,
The non-natural amino acid may be selected from the group consisting of L- (7-hydroxycoumarin-4-yl) ethylglycine, 3- (6-acetylnaphthalen-2-ylamino) -2-aminopropionic acid, Wherein the FRET sensor for detecting L-glutamine comprises an amino acid selected from the group consisting of combinations of &lt; RTI ID = 0.0 &gt;
The method according to claim 1,
The fluorescent receptor protein may be selected from the group consisting of green fluorescent protein (GFP), modified green fluorescent protein (mGFP), enhanced green fluorescent protein (eGFP), red fluorescent protein (RFP), modified red fluorescent protein (mRFP) A modified fluorescent substance (eIF), a fluorescent protein (eRFP), a blue fluorescent protein (BFP), a modified blue fluorescent protein (mBFP), an enhanced blue fluorescent protein (eBFP), a yellow fluorescent protein (YFP), a modified yellow fluorescent protein Wherein the protein is selected from the group consisting of protein (eYFP), blue fluorescent protein (CFP), modified blue fluorescent protein (mCFP), enhanced blue fluorescent protein (eCFP), and combinations thereof. FRET sensor for detection.
The method according to claim 1,
Wherein the L-glutamine-binding protein comprises SEQ ID NO: 2 and the fluorescent donor is introduced at the F136 or N138 position in the L-glutamine-binding protein of SEQ ID NO: 2.
The method according to claim 1,
Wherein said fluorescent receptor protein is linked to the N-terminus of said L-glutamine-binding protein.
A method for detecting L-glutamine using the FRET sensor for detecting L-glutamine according to any one of claims 1 to 6.
8. The method of claim 7,
Wherein the L-glutamine is detected by measuring the change in the FRET ratio of the FRET sensor according to the concentration of L-glutamine.
A composition for detecting L-glutamine, comprising a FRET sensor for detecting L-glutamine according to any one of claims 1 to 6.

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