Branched amino acid fluorescent probes and uses thereof

Abstract

The present application provides a fluorescent probe comprising a) a responsive polypeptide, and b) an optically active polypeptide, wherein the optically active polypeptide is inserted into the responsive polypeptide. The present application also provides a nucleic acid sequence encoding the fluorescent probe of any embodiment of the present application or its complement. The present application also provides an expression vector comprising the nucleic acid sequence of the present application or its complement operably linked to an expression control sequence. The present application also provides a cell comprising the expression vector of the present application. The present application also provides a method of producing the fluorescent probe of the present application, comprising: providing a cell comprising a vector expressing the fluorescent probe of the present application, culturing the cell under conditions in which the fluorescent probe is expressed by the cell, and isolating the fluorescent probe. The present application also provides the use of the fluorescent probe of the present application or the fluorescent probe produced by the method of the present application in detecting a branched chain amino acid. In one embodiment, the branched chain amino acid is selected from the group consisting of leucine, isoleucine and valine. The detection can be performed in vivo, in vitro, subcellularly or in situ. The present application also provides a kit comprising the fluorescent probe of the present application or the fluorescent probe produced by the method of the present application.

Description

Technical Field

[0001] This invention relates to fluorescent proteins, and more specifically to fluorescent probes for detecting branched-chain amino acids, their preparation methods, and applications. Background Technology

[0002] Branched-chain amino acids (BCAAs), including leucine, isoleucine, and valine, serve as effective nutrient signaling molecules that regulate protein synthesis rates. These three BCAAs are among the nine essential amino acids for humans, accounting for 35% of the essential amino acids in muscle protein and 40% of the amino acids required by mammals. They play a crucial role in immunity, brain function, and other physiological processes (PC Calder, 2006; B. Skeie et al., 1990). All three BCAAs are essential for lymphocyte growth and proliferation (K. Tajiri and Y. Shimizu, 2013). BCAAs may influence brain protein synthesis and energy production, and participate in the synthesis of various neurotransmitters (JD Fernstrom, 2005).

[0003] Leucine is the most important branched-chain amino acid (Buse and Reid, 1975; Buse et al., 1979; Li and Jefferson, 1978; Anthony et al., 2000). It can stimulate the initiation of mRNA translation through both insulin-dependent and independent pathways, thereby stimulating muscle protein synthesis (Norton and Layman, 2006). Leucine can cross the blood-brain barrier to some extent (Smith et al., 1987) and is more readily absorbed than other amino acids (Grill et al., 1992). Leucine plays a unique regulatory role in metabolism, including translational control of protein synthesis (Kimball and Jefferson, 2001) and blood glucose regulation (Layman and Baum, 2004). Studies have shown that abnormalities in branched-chain amino acid content are often associated with some major metabolic diseases, including diabetes (Hu, F. B et al., 2001; Wang, T. J et al., 2011; Mapstone, M et al., 2014), obesity (Goffredo et al., 2017), aging (D'Antona et al., 2010), and cancer (Tarlungeanu, D. C et al., 2016; Mayers, JR et al., 2014; Tonjes, M et al., 2013).

[0004] Because branched-chain amino acids, especially leucine, play such important roles, the detection of their content is particularly important. Commonly used detection methods include mass spectrometry (Pontoni, G et al., 2014, 1996), chromatography (Tateda N et al., Analytical Sciences: the international journal of the Japan Society for Analytical Chemistry 2001, 17(6):775-778; Waud S et al., Journal of chromatography B, Analytical technologies in the biomedical and life sciences 2002, 767(2):369-374), capillary electrophoresis (Li Xt et al., Chem Res Chin Univ 2013, 29(3):434-438; Meng J et al., The Analyst 2010, 135(7):1592-1599), and ultraviolet-visible spectrophotometry (Hortala MA et al., J AmChem Soc 2003, 125(1):20-21; Pu F et al., Anal Chem 2010, 82(19):8211-8216; Du J et al., Chemical Communications (Cambridge, England) 2013, 49(47):5399-5401; Engeser M et al., Chemical Communications 1999, (13):1191-1192). However, these detection methods have significant technical limitations, requiring cell disruption, time-consuming sample processing, and difficulties in separation, extraction, and purification, making it impossible to perform high-throughput quantitative analysis in situ, which to some extent restricts the development of research in the field of branched-chain amino acids.

[0005] Therefore, there is an urgent need in this field to develop a highly specific branched-chain amino acid detection technology, especially a detection method suitable for both physiological and subcellular levels that is in situ, real-time, dynamic, high-throughput, and has high spatiotemporal resolution.

[0006] Branched-chain amino acids have also been determined in this field by fluorescence spectroscopy (Engeser M et al., Chemical Communications 1999,(13):1191-1192), which measures the change in fluorescence emission intensity of naphthalene fragments covalently linked to Ni(II) tetraazamacyclocyclic complexes. However, the Ni(II) tetraazamacyclocyclic complexes require temperature-dependent spin exchange equilibrium. This method still cannot meet the above monitoring requirements.

[0007] Compared to traditional small molecule dye detection technology and rapidly developing quantum dot detection technology, fluorescent protein detection technology has a unique and overwhelming advantage in most live cell imaging applications. It can be genetically introduced into cells, tissues, and even entire organs. Therefore, fluorescent proteins can serve as whole-cell markers or indicators of gene activation.

[0008] Green fluorescent protein was originally extracted from the Victoria victoria. Wild-type AvGFP consists of 238 amino acids with a molecular weight of approximately 26 kD. Current research confirms that the three amino acids Ser-Tyr-Gly at positions 65-67 of the natural GFP protein can spontaneously form a fluorescent chromophore: p-hydroxybenzylideneimidazolinone, which is its main luminescent site. The spectral characteristics of wild-type AvGFP are quite complex. Its main fluorescence excitation peak is at 395 nm, while there is another secondary peak at 475 nm, the latter having a fluorescence intensity of about 1 / 3 of the former. Under standard solution conditions, excitation at 395 nm can produce emission at 508 nm, while excitation at 475 nm produces a maximum emission wavelength at 503 nm (Heim, R. et al., Proc Natl Acad Sci US A. 1994, V. 91(26), pp. 12501-12504).

[0009] With the deepening research on GFP protein mutations, various high-performance GFP derivatives have been developed using molecular biology techniques. By performing different single-point mutations or combinations on wild-type GFP, enhanced GFP (S65T, F64L), YFP (T203Y), and CFP (Y66W) can be obtained. By rearranging the GFP protein sequence, the original amino acid 145-238 is used as the N-terminus of the new protein, and the original amino acid 1-144 is used as the C-terminus of the new protein. The two fragments are connected by a short, flexible peptide chain, forming a circularly permuted fluorescent protein that is sensitive to spatial changes. Based on this, a point mutation of the original protein T203Y forms the circularly permuted yellow fluorescent protein cpYFP (Nagai, T. et al., Proc Natl Acad Sci US A.2001, V.98(6), pp.3197-3202).

[0010] As research into fluorescent proteins deepens, related fluorescence-based analytical detection techniques have also been further developed. For example, the commonly used fluorescence resonance energy transfer (FRET) technique works on the principle that when two fluorescent chromophores are sufficiently close, the donor molecule absorbs photons of a certain frequency and is excited to a higher electronic energy state. Before this electron returns to its ground state, energy is transferred to the neighboring acceptor molecule through dipole interactions (i.e., energy resonance transfer occurs). FRET is a non-radiative energy transition that transfers energy from the excited state of the donor to the excited state of the acceptor through intermolecular electric dipole interactions. This reduces the fluorescence intensity of the donor, while the acceptor may emit a stronger characteristic fluorescence (sensitized fluorescence) or no fluorescence (fluorescence quenching). Further research on green fluorescent protein has revealed that cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), derived from mutants of green fluorescent protein, form a high-performing donor / acceptor pair. The emission spectrum of CFP and the absorption spectrum of YFP have considerable overlap. When they are sufficiently close, excitation with the absorption wavelength of CFP will cause the chromophore of CFP to resonantly transfer energy efficiently to the chromophore of YFP. Therefore, the fluorescence emission of CFP will be weakened or disappear, and the fluorescence of YFP will become the dominant emission. The energy conversion efficiency between the two chromophores is inversely proportional to the sixth power of the spatial distance between them, making it highly sensitive to changes in spatial position. Therefore, current research reports the use of genetic engineering recombination techniques to fuse the ends of the gene of the protein to be studied with CFP and YFP respectively, expressing a novel fusion protein. The spatial changes resulting from the binding of this protein to its specific target molecule are visually manifested through changes in fluorescence.

[0011] Therefore, the fluorescent protein sequences used in this paper can be derived from fluorescent proteins and their derivatives of Aequoreavictoria, including but not limited to the sequences of these mutants: yellow fluorescent protein (YFP), green fluorescent protein (GFP), cyan fluorescent protein (CFP), etc., with the sequence of yellow fluorescent protein YFP being preferred, and the sequence of circularly arranged yellow fluorescent protein cpYFP being more preferred.

[0012] Both branched-chain amino acid and leucine-binding proteins are derived from Escherichia coli or Salmonella. Branched-chain amino acid and leucine-binding proteins share more than 90% homology with each other. They contain the typical structure of periplasmic binding proteins, where two α / β globular domains are connected by a hinge, and can bind branched-chain amino acids and leucine.

[0013] The inventors discovered that a fluorescent probe fused with a fluorescent protein and a branched-chain amino acid-binding protein or a leucine-binding protein can display the binding of the branched-chain amino acid-binding protein or the leucine-binding protein to the branched-chain amino acid or leucine in a real-time and intuitive manner.

[0014] References cited or discussed herein should not be construed as an admission that such references are prior art of the present invention. Summary of the Invention

[0015] The purpose of this invention is to provide a fluorescent probe for branched-chain amino acids or leucine that can be located, detected in real time, and with high throughput and quantification inside and outside cells.

[0016] This invention provides a fluorescent probe comprising a) a leucine-responsive polypeptide and b) an optically active polypeptide, wherein the optically active polypeptide is inserted into the leucine-responsive polypeptide. In one embodiment, the leucine-responsive polypeptide also responds to other branched-chain amino acids, such as isoleucine or valine.

[0017] In one embodiment, the optically active polypeptide is a fluorescent protein or a functional fragment or variant thereof. In one embodiment, the fluorescent protein is selected from green fluorescent protein, blue fluorescent protein, cyan fluorescent protein, orange fluorescent protein, apple red fluorescent protein, and red fluorescent protein. In one embodiment, the fluorescent protein has the sequence shown in SEQ ID NO: 3-13.

[0018] In one embodiment, the fluorescent probe of the present invention further comprises one or more linkers between a leucine-responsive polypeptide and an optically active polypeptide. In one embodiment, the fluorescent probe of the present invention does not contain a linker. The linker of the present invention can be any amino acid sequence, preferably no longer than 4 amino acids.

[0019] In one embodiment, the fluorescent probe of the present invention further comprises a localization sequence for positioning the probe to, for example, a specific organelle within a cell.

[0020] In one embodiment, the leucine-responsive polypeptide of the present invention is selected from branched-chain amino acid-binding proteins or functional fragments thereof, and leucine-binding proteins or functional fragments thereof.

[0021] In one embodiment, the leucine-responsive polypeptide of the present invention is a branched-chain amino acid-binding protein or a functional fragment thereof, preferably having the sequence shown in SEQ ID NO: 1 or a functional fragment thereof, or a sequence having 85% sequence identity with it.

[0022] In one embodiment, the leucine-responsive polypeptide of the present invention is a leucine-binding protein or a functional fragment thereof, preferably having the sequence shown in SEQ ID NO: 2 or a functional fragment thereof, or a sequence having 85% sequence identity with it.

[0023] The optically active peptide of this invention can be located at any position in the leucine-responsive peptide. In one embodiment, the optically active peptide of this invention is located between residues 118-120, 248-258, or 325-331 of the leucine-responsive peptide, with the numbering corresponding to the full length of the branched-chain amino acid-binding protein or the full length of the leucine-binding protein. In one embodiment, the optically active peptide replaces one or more amino acids between residues 118-120, 248-258, or 325-331 of the leucine-responsive peptide, with the numbering corresponding to the full length of the branched-chain amino acid-binding protein or the full length of the leucine-binding protein.

[0024] In one embodiment, the optically active polypeptide is inserted into the following sites of the branched-chain amino acid-binding protein: 118 / 119, 118 / 120, 119 / 120, 248 / 249, 248 / 250, 248 / 251, 248 / 252, 248 / 253, 248 / 254, 248 / 255, 248 / 256, 248 / 257, 248 / 258, 249 / 250. 249 / 251, 249 / 252, 249 / 253, 249 / 254, 249 / 255, 249 / 256, 249 / 257, 249 / 258, 250 / 251, 250 / 252, 250 / 253, 250 / 254, 250 / 255, 250 / 256, 250 / 257, 250 / 258, 251 / 252, 251 / 253 251 / 254, 251 / 255, 251 / 256, 251 / 257, 251 / 258, 252 / 253, 252 / 254, 252 / 255, 252 / 256, 252 / 257, 252 / 258, 253 / 254, 253 / 255, 253 / 256, 253 / 257, 253 / 258, 254 / 255, 254 / 256 254 / 257, 254 / 258, 255 / 256, 255 / 257, 255 / 258, 256 / 257, 256 / 258, 257 / 258, 325 / 326, 325 / 327, 325 / 328, 325 / 329, 326 / 327, 326 / 328, 326 / 329, 327 / 328, 327 / 329 or 328 / 329.

[0025] In one embodiment, the optically active polypeptide is inserted into the following sites of the leucine-binding protein: 118 / 119, 118 / 120, 119 / 120, 248 / 249, 248 / 250, 248 / 251, 248 / 252, 248 / 253, 248 / 254, 248 / 255, 248 / 256, 248 / 257, 248 / 258, 249 / 250, 2 49 / 251, 249 / 252, 249 / 253, 249 / 254, 249 / 255, 249 / 256, 249 / 257, 249 / 258, 250 / 251, 250 / 252, 250 / 253, 250 / 254, 250 / 255, 250 / 256, 250 / 257, 250 / 258, 251 / 252, 251 / 253, 2 51 / 254, 251 / 255, 251 / 256, 251 / 257, 251 / 258, 252 / 253, 252 / 254, 252 / 255, 252 / 256, 252 / 257, 252 / 258, 253 / 254, 253 / 255, 253 / 256, 253 / 257, 253 / 258, 254 / 255, 254 / 256 254 / 257, 254 / 258, 255 / 256, 255 / 257, 255 / 258, 256 / 257, 256 / 258, 257 / 258, 327 / 328, 327 / 329, 327 / 330, 327 / 331, 328 / 329, 328 / 330, 328 / 331, 329 / 330, 329 / 331 or 330 / 331.

[0026] In one embodiment, the optically active peptide is located between residues 325-329 of the leucine-responsive peptide, with the numbering corresponding to the full length of the branched-chain amino acid-binding protein. In another embodiment, the optically active peptide replaces one or more amino acids between residues 325-329 of the leucine-responsive peptide, with the numbering corresponding to the full length of the branched-chain amino acid-binding protein. Preferably, the optically active peptide is located between residues 326 / 327 or 327 / 328 of the branched-chain amino acid-binding protein or a functional fragment thereof.

[0027] In one embodiment, the optically active peptide is located between residues 327-331 of the leucine-responsive peptide, with the numbering corresponding to the full length of the leucine-binding protein. In another embodiment, the optically active peptide replaces one or more amino acids between residues 327-331 of the leucine-responsive peptide, with the numbering corresponding to the full length of the leucine-binding protein. Preferably, the optically active peptide is located between residues 328 / 329 or 329 / 330 of the leucine-binding protein or a functional fragment thereof.

[0028] In another aspect, the present invention provides a fluorescent probe comprising a) a polypeptide responsive to branched-chain amino acids, and b) an optically active polypeptide, wherein the optically active polypeptide is inserted into the polypeptide responsive to branched-chain amino acids.

[0029] In one embodiment, the optically active polypeptide is a fluorescent protein or a functional fragment or variant thereof. In one embodiment, the fluorescent protein is selected from green fluorescent protein, blue fluorescent protein, cyan fluorescent protein, orange fluorescent protein, apple red fluorescent protein, and red fluorescent protein. In one embodiment, the fluorescent protein has the sequence shown in SEQ ID NO: 3-13.

[0030] In one embodiment, the fluorescent probe includes one or more linkers between a peptide that responds to branched-chain amino acids and an optically active peptide. In another embodiment, the fluorescent probe does not include a linker. The linker described in this invention can be any amino acid sequence, preferably no longer than four amino acids.

[0031] In one embodiment, the fluorescent probe includes a localization sequence for targeting the probe to a specific organelle, such as that of a cell.

[0032] In one embodiment, the branched-chain amino acid is selected from leucine, isoleucine, and valine.

[0033] In one embodiment, the polypeptide responding to branched-chain amino acids is selected from branched-chain amino acid-binding proteins or functional fragments thereof, and leucine-binding proteins or functional fragments thereof.

[0034] In one embodiment, the polypeptide responding to branched-chain amino acids is a branched-chain amino acid-binding protein or a functional fragment thereof, preferably having the sequence shown in SEQ ID NO: 1 or a functional fragment thereof, or a sequence having 85% sequence identity with it.

[0035] In one embodiment, the polypeptide responding to branched-chain amino acids is a leucine-binding protein or a functional fragment thereof, preferably having the sequence shown in SEQ ID NO: 2 or a functional fragment thereof, or a sequence having 85% sequence identity with it.

[0036] In this invention, the optically active peptide can be located at any position in the branched-chain amino acid-responsive peptide. In one embodiment, the optically active peptide is located between residues 118-120, 248-258, or 325-331 of the branched-chain amino acid-responsive peptide, with the numbering corresponding to the full length of the branched-chain amino acid-binding protein or the full length of the leucine-binding protein. In one embodiment, the optically active peptide replaces one or more amino acids between residues 118-120, 248-258, or 325-331 of the branched-chain amino acid-responsive peptide, with the numbering corresponding to the full length of the branched-chain amino acid-binding protein or the full length of the leucine-binding protein.

[0037] In one embodiment, the optically active polypeptide of the present invention inserts into the following sites of a branched-chain amino acid-binding protein: 118 / 119, 118 / 120, 119 / 120, 248 / 249, 248 / 250, 248 / 251, 248 / 252, 248 / 253, 248 / 254, 248 / 255, 248 / 256, 248 / 257, 248 / 258, 249 / 250, 249 / 251, 249 / 252, 249 / 253, 249 / 254, 249 / 255, 249 / 256, 249 / 257, 249 / 258, 250 / 251, 250 / 252, 250 / 253, 250 / 254, 250 / 255, 250 / 256, 250 / 257, 250 / 258, 251 / 252, 251 / 2 53, 251 / 254, 251 / 255, 251 / 256, 251 / 257, 251 / 258, 252 / 253, 252 / 254, 252 / 255, 252 / 256, 252 / 257, 252 / 258, 253 / 254, 253 / 255, 253 / 256, 253 / 257, 253 / 258, 254 / 255, 254 / 25 6, 254 / 257, 254 / 258, 255 / 256, 255 / 257, 255 / 258, 256 / 257, 256 / 258, 257 / 258, 325 / 326, 325 / 327, 325 / 328, 325 / 329, 326 / 327, 326 / 328, 326 / 329, 327 / 328, 327 / 329, or 328 / 329. In this text, if two numbers in a site represented as “X / Y” are not consecutive, it indicates that the optically active peptide has replaced the amino acid between those numbers. For example, 250 / 257 indicates that the optically active peptide has replaced amino acid sequences 251-256.

[0038] In one embodiment, the optically active polypeptide of the present invention inserts into the following sites of the leucine-binding protein: 118 / 119, 118 / 120, 119 / 120, 248 / 249, 248 / 250, 248 / 251, 248 / 252, 248 / 253, 248 / 254, 248 / 255, 248 / 256, 248 / 257, 248 / 258, 249 / 2 50, 249 / 251, 249 / 252, 249 / 253, 249 / 254, 249 / 255, 249 / 256, 249 / 257, 249 / 258, 250 / 251, 250 / 252, 250 / 253, 250 / 254, 250 / 255, 250 / 256, 250 / 257, 250 / 258, 251 / 252, 251 / 25 3, 251 / 254, 251 / 255, 251 / 256, 251 / 257, 251 / 258, 252 / 253, 252 / 254, 252 / 255, 252 / 256, 252 / 257, 252 / 258, 253 / 254, 253 / 255, 253 / 256, 253 / 257, 253 / 258, 254 / 255, 254 / 256 The sequences are: 254 / 257, 254 / 258, 255 / 256, 255 / 257, 255 / 258, 256 / 257, 256 / 258, 257 / 258, 327 / 328, 327 / 329, 327 / 330, 327 / 331, 328 / 329, 328 / 330, 328 / 331, 329 / 330, 329 / 331, or 330 / 331. In this text, if two numbers in a site represented as “X / Y” are not consecutive, it indicates that the optically active peptide has replaced the amino acid between those numbers. For example, 250 / 257 indicates that the optically active peptide has replaced amino acid sequences 251-256.

[0039] In one embodiment, the optically active polypeptide of the present invention is located between residues 325-329 of a branched-chain amino acid-binding protein or its functional fragment, with the numbering corresponding to the full length of the branched-chain amino acid-binding protein. Preferably, the optically active polypeptide of the present invention is located between residues 326 / 327 or 327 / 328 of a branched-chain amino acid-binding protein or its functional fragment. In one embodiment, the optically active polypeptide of the present invention replaces one or more amino acids between residues 325-329 of a branched-chain amino acid-binding protein or its functional fragment, with the numbering corresponding to the full length of the branched-chain amino acid-binding protein.

[0040] In one embodiment, the optically active polypeptide of the present invention is located between residues 327-331 of the leucine-binding protein or its functional fragment, with the numbering corresponding to the full length of the leucine-binding protein. In another embodiment, the optically active polypeptide of the present invention replaces one or more amino acids between residues 327-331 of the leucine-binding protein or its functional fragment, with the numbering corresponding to the full length of the leucine-binding protein. Preferably, the optically active polypeptide of the present invention is located between residues 328 / 329 or 329 / 330 of the leucine-binding protein or its functional fragment.

[0041] The present invention also provides a nucleic acid sequence encoding the fluorescent probe described herein or its complementary sequence.

[0042] The present invention also provides an expression vector comprising the nucleic acid sequence of the present invention or its complementary sequence thereof operatively linked to an expression control sequence, the nucleic acid sequence encoding the fluorescent probe of the present invention.

[0043] The present invention also provides cells comprising the expression vector of the present invention, wherein the expression vector comprises the nucleic acid sequence of the present invention or its complementary sequence thereof operatively linked to an expression control sequence.

[0044] The present invention provides a method for preparing the fluorescent probe of the present invention, comprising: providing a cell containing a vector expressing the fluorescent probe of the present invention, culturing the cell under conditions of cell expression, and isolating the fluorescent probe.

[0045] The present invention provides a method for detecting leucine in a sample, comprising: contacting the sample with a fluorescent probe as described in the present invention or a fluorescent probe prepared by the method of the present invention, and detecting changes in optically active peptides.

[0046] This invention provides a method for detecting branched-chain amino acids in a sample, comprising: contacting the sample with a fluorescent probe described in this invention or a fluorescent probe prepared by the method of this invention, and detecting changes in optically active peptides. In one embodiment, the branched-chain amino acids are selected from leucine, isoleucine, and valine. The detection can be performed in vivo, in vitro, subcellular, or in situ.

[0047] This invention provides the use of the fluorescent probe described herein or the fluorescent probe prepared by the method of this invention in the detection of leucine in a sample. The detection can be performed in vivo, in vitro, subcellular, or in situ.

[0048] This invention provides the use of the fluorescent probe described herein or the fluorescent probe prepared by the method of this invention in the detection of branched-chain amino acids in a sample. In one embodiment, the branched-chain amino acids are selected from leucine, isoleucine, and valine. The detection can be performed in vivo, in vitro, subcellular, or in situ.

[0049] The present invention also provides a kit containing the fluorescent probe described in the present invention or the fluorescent probe prepared by the method of the present invention.

[0050] The beneficial effects of this invention are as follows: The fluorescent probes described in this invention are easy to mature, exhibit large fluorescence dynamics, and have good specificity. Furthermore, they can be expressed in cells through gene manipulation, enabling real-time, high-throughput, and quantitative detection of branched-chain amino acids and leucine both inside and outside the cell. This eliminates the time-consuming sample processing steps. Experimental results show that the branched-chain amino acid and leucine fluorescent probes provided in this application achieve a maximum response of more than 3 times to branched-chain amino acids and leucine, allowing for localized detection in subcellular structures such as the cytoplasm, mitochondria, nucleus, endoplasmic reticulum, outer membrane, inner membrane, Golgi apparatus, and lysosomes; and enabling high-throughput compound screening. Attached Figure Description

[0051] Figure 1 The above are SDS-PAGE analysis images of the branched-chain amino acid fluorescent probe and the leucine fluorescent probe described in the examples. Figure 2 This is a graph showing the response changes of the yellow fluorescent protein cpYFP described in Example 1 to branched-chain amino acid fluorescent probes and leucine fluorescent probes formed at different insertion sites in branched-chain amino acid binding proteins and leucine binding proteins. Figure 3 This is a graph showing the response changes of the blue fluorescent protein cpBFP described in Example 2 to branched-chain amino acid fluorescent probes and leucine fluorescent probes formed at different insertion sites in branched-chain amino acid binding proteins and leucine binding proteins. Figure 4 This is a graph showing the response changes of the branched-chain amino acid fluorescent probes and leucine fluorescent probes formed by the apple red fluorescent protein cpmApple at different insertion sites of branched-chain amino acid binding proteins and leucine binding proteins as described in Example 3. Figure 5 This demonstrates the response of fusion proteins formed by linking the three fluorescent proteins described in Example 4 to the N-terminus or C-terminus of branched-chain amino acid fluorescent probes or leucine fluorescent probes to branched-chain amino acids. For example... Figure 5 The fusion protein showed no statistically significant change compared to the control.

[0052] Figure 6 The fluorescence spectra of the branched-chain amino acid fluorescent probe and the leucine fluorescent probe described in Example 5 are shown in the diagram. Figure 7 The titration curves of the branched-chain amino acid fluorescent probe described in Example 5 for different concentrations of branched-chain amino acids and the titration curves of the leucine fluorescent probe described in Example 5 for different concentrations of leucine or branched-chain amino acids. Figure 8To illustrate the localization and distribution of the branched-chain amino acid fluorescent probes in subcellular organelles of mammalian cells as described in the six examples; Figure 9 The branched-chain amino acid fluorescent probe described in Example 6 was used to dynamically monitor the transmembrane transport of branched-chain amino acids in different subcellular organelles of mammalian cells. Figure 10 This is a diagram illustrating the high-throughput compound screening analysis based on branched-chain amino acid fluorescent probes at the live cell level as described in Example 7. Figure 11 This is a graph showing the quantitative analysis of branched-chain amino acid fluorescent probes in culture medium and blood as described in Example 8. Detailed Implementation

[0053] I. Definition: When a value or range is given, the term “about” as used herein means that the value or range is within 20%, 10%, and 5% of the given value or range.

[0054] The terms “comprising,” “including,” and their equivalents as used herein include the meanings of “containing” and “composed of,” for example, a composition “comprising” X may consist of only X or may contain other substances, such as X+Y.

[0055] As used in this article, "branched-chain amino acid" or "BCAA" refers to an amino acid with aliphatic side chains (branched to a central carbon atom with three or more atoms). Among protein amino acids, there are three types of branched-chain amino acids: leucine, isoleucine, and valine. Non-protein branched-chain amino acids include 2-aminoisobutyric acid.

[0056] As used herein, the term "branched-chain amino acid-responsive peptide" refers to a peptide that responds to branched-chain amino acids. The term "leucine-responsive peptide" refers to a peptide that responds to leucine. Both may be collectively referred to as "responsive peptides." The response includes any response to chemical, biological, electrical, or physiological parameters of the peptide in relation to the interaction with the responsive peptide. Responses include small changes, such as changes in the orientation of amino acids or peptide fragments of the responsive peptide, and changes in, for example, the primary, secondary, or tertiary structure of the peptide, including, for example, changes in protonation, electrochemical potential, and / or conformation. "Conformation" is the three-dimensional arrangement of the primary, secondary, and tertiary structures of a molecule containing side groups; a conformational change occurs when the three-dimensional structure of the molecule changes. Examples of conformational changes include a change from an α-helix to a β-sheet or vice versa. It is understood that a detectable change need not be a conformational change, as long as the fluorescence of the fluorescent protein moiety is altered.

[0057] The branched-chain amino acid-responsive polypeptides or leucine-responsive polypeptides described in this invention include, but are not limited to, "proteins that bind to branched-chain amino acids," "branched-chain amino acid-binding proteins," "leucine-binding proteins," and "leucine-binding proteins." The exemplary branched-chain amino acid-binding protein LivJ or leucine-binding protein LivK described in this invention are derived from *Escherichia coli*, or from *Salmonella*, and are branched-chain amino acid-binding proteins or leucine-binding proteins with more than 90% homology to these proteins. They contain the typical structure of periplasmic binding proteins, with two α / β globular domains connected by a hinge, and can bind branched-chain amino acids or leucine. These branched-chain amino acid-binding proteins or leucine-binding proteins can sense changes in the concentration of branched-chain amino acids and leucine in the periplasm. During the dynamic changes in the concentration of branched-chain amino acids and leucine, the spatial conformation of these proteins also undergoes significant changes. Branched-chain amino acid (BCAA)-binding proteins and leucine-binding proteins specifically bind to physiological concentrations of BCAAs and leucine, resulting in conformational changes in fluorescent proteins. This leads to alterations in the fluorescence of the fluorescent proteins. Standard curves are plotted using fluorescence measurements at different BCAA and leucine concentrations to detect and analyze the presence and / or levels of BCAAs and leucine. An exemplary BCAA-binding protein is shown in SEQ ID NO. 1, and the amino acid sequence of an exemplary leucine-binding protein is shown in SEQ ID NO. 2.

[0058] As used herein, the term "fusion protein" is synonymous with "fluorescent fusion protein" and "recombinant fluorescent fusion protein," referring to a polypeptide or protein comprising the amino acid sequence of a first polypeptide or protein or a fragment thereof, analogue, or derivative thereof, and the amino acid sequence of a heterologous polypeptide or protein (i.e., a second polypeptide or protein or a fragment thereof, analogue, or derivative thereof, different from the first polypeptide or protein or a fragment thereof, analogue, or derivative thereof). In one embodiment, the fusion protein comprises a fluorescent protein fused to a heterologous protein, polypeptide, or peptide. According to this embodiment, the heterologous protein, polypeptide, or peptide may or may not be a different type of fluorescent protein. In one embodiment, the fusion protein retains or enhances activity compared to the original polypeptide or protein prior to fusion with the heterologous protein, polypeptide, or peptide. In a specific embodiment, the fusion protein comprises a fluorescent probe fused to a heterologous protein, polypeptide, or peptide, which may be a specific subcellular localization signal.

[0059] As used herein, the term "fluorescent probe" refers to a responsive polypeptide fused to a fluorescent protein. The responsive polypeptide may be a polypeptide responsive to branched-chain amino acids or a polypeptide responsive to leucine, specifically a branched-chain amino acid-binding protein or a leucine-binding protein. The fluorescent probe utilizes the conformational change in the fluorescent protein caused by the binding of the responsive polypeptide to a branched-chain amino acid (e.g., selected from leucine, isoleucine, and valine), thereby resulting in the generation or disappearance of fluorescence or a change in the generated fluorescence, to achieve the detection of the presence and / or level of the branched-chain amino acid.

[0060] In the fluorescent probes of this invention, an optically active polypeptide (e.g., a fluorescent protein) is operatively inserted into a responsive polypeptide, which may be a polypeptide responsive to branched-chain amino acids or a polypeptide responsive to leucine. A protein-based "optically active polypeptide" is a polypeptide containing a luminescent mechanism. Fluorescence is an optical property of an optically active polypeptide, which can be used as a means of detecting the responsiveness of the fluorescent probes or responsive polypeptides of this invention. As used herein, the term "fluorescence property" refers to the molar extinction coefficient at an appropriate excitation wavelength, fluorescence quantum efficiency, shape of the excitation or emission spectrum, maximum excitation wavelength and maximum emission wavelength, excitation ratio of amplitudes at two different wavelengths, emission amplitude ratio at two different wavelengths, excited-state lifetime, or fluorescence anisotropy.

[0061] Measurable differences in any of these properties between the active and inactive states are sufficient for the utility of the fluorescent protein substrates of the present invention in activity assays. Measurable differences can be determined by determining the amount of any quantitative fluorescence property, such as the amount of fluorescence at a specific wavelength or the integral of fluorescence over the emission spectrum. Preferably, the protein substrate is selected to have fluorescence properties that are easily distinguishable between inactive and activated conformational states.

[0062] As used herein, the term "fluorophore" is synonymous with "fluorescent protein," referring to proteins that fluoresce on their own or under illumination. Fluorescent proteins are commonly used in detection methods, such as the green fluorescent protein GFP, which is frequently used in the biotechnology field, and its mutant derivatives BFP, CFP, YFP, and cpYFP. Any fluorescent protein can be used in this invention, including proteins that fluoresce due to intramolecular rearrangement or those with added cofactors that promote fluorescence. The sequences of exemplary fluorescent proteins are shown in SEQ ID NO:3-13.

[0063] The term "GFP" as used in this article refers to green fluorescent protein, originally derived from the bioluminescent jellyfish *Epipremnum aureum*. Aequorea victoria Extracted from wild-type AvGFP consists of 238 amino acids with a molecular weight of approximately 26 kDa. GFP has a unique barrel-shaped structure formed by 12 β-sheet chains, encapsulating a chromophore tripeptide (Ser65-Tyr66-Gly67). In the presence of oxygen, it spontaneously forms a chromophore structure of p-hydroxybenzylimidazolinone, producing fluorescence. GFP fluorescence does not require cofactors and is very stable, making it an excellent imaging tool. Current research confirms that the three amino acids Ser-Tyr-Gly at positions 65-67 of the native GFP protein can spontaneously form a fluorescent chromophore: p-hydroxybenzylimidazolinone (…). p -hydroxybenzylideneimidazolinone) is its main luminescent site. Wild type Av The spectral characteristics of GFP are quite complex. Its main fluorescence excitation peak is at 395 nm, while there is a secondary peak at 475 nm, the latter having an amplitude intensity of about 1 / 3 that of the former. Under standard solution conditions, excitation at 395 nm can produce emission at 508 nm, while excitation at 475 nm produces a maximum emission wavelength at 503 nm.

[0064] The term "YFP" used in this article refers to yellow fluorescent protein, which is derived from green fluorescent protein (GFP). Its amino acid sequence shares over 90% homology with GFP. The key difference between YFP and GFP lies in the mutation of amino acid position 203 from threonine to tyrosine (T203Y). Compared to the original AvGFP, the main excitation wavelength of YFP is redshifted to 514 nm, while the emission wavelength changes to 527 nm. Based on this, a site-directed mutation (S65T) at amino acid position 65 of YFP yields the fluorescence-enhanced yellow fluorescent protein EYFP.

[0065] The fluorescent protein of this invention can be a yellow fluorescent protein cpYFP, ​​the amino acid sequence of which is shown in SEQ ID NO.3. In this invention, the yellow fluorescent protein cpYFP is obtained by connecting the original N-terminus and C-terminus of GFP with a flexible short peptide chain, creating a new N-terminus and C-terminus at the chromophore position of the original GFP. The original amino acid positions 145-238 are used as the N-terminus of the new protein, and the original amino acid positions 1-144 are used as the C-terminus. The two fragments are connected by 5-9 flexible short peptide chains, forming a spatially sensitive circularly arranged yellow fluorescent protein cpYFP (circular permutation yellow fluorescent protein). In this invention, the chromophore position is preferably at amino acids Y144 and N145; the flexible short peptide chain is preferably VDGGSGGTG or GGSGG.

[0066] In this invention, the red fluorescent protein cpmKate was originally extracted from corals in the ocean. Wild RFP is an oligomeric protein that is not conducive to the fusion expression of organisms. Subsequently, red fluorescent proteins of different color bands were further derived based on RFP, among which mCherry and mKate are the most commonly used.

[0067] In other embodiments of the present invention, the fluorescent protein may also be one or more of the following: green fluorescent protein with an amino acid sequence as shown in SEQ ID NO. 6 or SEQ ID NO. 11; blue fluorescent protein with an amino acid sequence as shown in SEQ ID NO. 4 or SEQ ID NO. 12; cyan fluorescent protein cpTFP with an amino acid sequence as shown in SEQ ID NO. 7; orange fluorescent protein with an amino acid sequence as shown in SEQ ID NO. 8; apple red fluorescent protein with an amino acid sequence as shown in SEQ ID NO. 5; red fluorescent protein cpmKate with an amino acid sequence as shown in SEQ ID NO. 9 or SEQ ID NO. 13; and red fluorescent protein mcherry with an amino acid sequence as shown in SEQ ID NO. 10.

[0068] In the fluorescent probe of this invention, the fluorescent protein or its functional fragment or variant can be located at any position of the responsive polypeptide, which can be a polypeptide responding to branched-chain amino acids or a polypeptide responding to leucine. Specifically, the fluorescent protein or its functional fragment or variant is located in a flexible region of the responsive polypeptide. This flexible region refers to specific structures, such as ring domains, present in the higher-order structure of a protein. These domains have higher mobility and flexibility compared to other higher-order structures of the protein, and their spatial conformation can dynamically change after the protein binds to the ligand. The flexible region described in this invention can be the region where the insertion site is located in branched-chain amino acid-binding proteins and leucine-binding proteins. In one embodiment, the fluorescent protein of the present invention, or a functional fragment or variant thereof, is located between residues 118-120, 248-258, or 325-331 of the responsive peptide, with the numbering corresponding to the full length of the branched-chain amino acid-binding protein or the full length of the leucine-binding protein. In another embodiment, the fluorescent protein of the present invention, or a functional fragment or variant thereof, replaces one or more amino acids between residues 118-120, 248-258, or 325-331 of the responsive peptide, with the numbering corresponding to the full length of the branched-chain amino acid-binding protein or the full length of the leucine-binding protein. In one embodiment, the fluorescent protein of the present invention, or a functional fragment or variant thereof, is inserted into the following sites of a branched-chain amino acid-binding protein: 118 / 119, 118 / 120, 119 / 120, 248 / 249, 248 / 250, 248 / 251, 248 / 252, 248 / 253, 248 / 254, 248 / 255, 248 / 256, 248 / 257, 248 / 258. 249 / 250, 249 / 251, 249 / 252, 249 / 253, 249 / 254, 249 / 255, 249 / 256, 249 / 257, 249 / 258, 250 / 251, 250 / 252, 250 / 253, 250 / 254, 250 / 255, 250 / 256, 250 / 257, 250 / 258, 251 / 252, 25 1 / 253, 251 / 254, 251 / 255, 251 / 256, 251 / 257, 251 / 258, 252 / 253, 252 / 254, 252 / 255, 252 / 256, 252 / 257, 252 / 258, 253 / 254, 253 / 255, 253 / 256, 253 / 257, 253 / 258, 254 / 255, 254 / 2 56, 254 / 257, 254 / 258, 255 / 256, 255 / 257, 255 / 258, 256 / 257, 256 / 258, 257 / 258, 325 / 326, 325 / 327, 325 / 328, 325 / 329, 326 / 327, 326 / 328, 326 / 329, 327 / 328, 327 / 329 or 328 / 329.In one embodiment, the fluorescent protein of the present invention, or a functional fragment or variant thereof, is inserted into the following sites of a leucine-binding protein: 118 / 119, 118 / 120, 119 / 120, 248 / 249, 248 / 250, 248 / 251, 248 / 252, 248 / 253, 248 / 254, 248 / 255, 248 / 256, 248 / 257, 248 / 258. 249 / 250, 249 / 251, 249 / 252, 249 / 253, 249 / 254, 249 / 255, 249 / 256, 249 / 257, 249 / 258, 250 / 251, 250 / 252, 250 / 253, 250 / 254, 250 / 255, 250 / 256, 250 / 257, 250 / 258, 251 / 252, 251 / 253, 251 / 254, 251 / 255, 251 / 256, 251 / 257, 251 / 258, 252 / 253, 252 / 254, 252 / 255, 252 / 256, 252 / 257, 252 / 258, 253 / 254, 253 / 255, 253 / 256, 253 / 257, 253 / 258, 254 / 255, 254 / 2 56, 254 / 257, 254 / 258, 255 / 256, 255 / 257, 255 / 258, 256 / 257, 256 / 258, 257 / 258, 327 / 328, 327 / 329, 327 / 330, 327 / 331, 328 / 329, 328 / 330, 328 / 331, 329 / 330, 329 / 331, or 330 / 331. In one embodiment, the fluorescent protein or its functional fragment or variant thereof of the present invention is located between residues 325-329 of the branched-chain amino acid binding protein or its functional fragment, and the numbering corresponds to the full length of the branched-chain amino acid binding protein. In one embodiment, the fluorescent protein or its functional fragment or variant thereof of the present invention replaces one or more amino acids between residues 325-329 of the branched-chain amino acid binding protein or its functional fragment, and the numbering corresponds to the full length of the branched-chain amino acid binding protein. Preferably, the fluorescent protein or its functional fragment or variant thereof of the present invention is located between residues 326 / 327 or 327 / 328 of the branched-chain amino acid-binding protein or its functional fragment. In one embodiment, the fluorescent protein or its functional fragment or variant thereof of the present invention is located between residues 327-331 of the leucine-binding protein or its functional fragment, with the numbering corresponding to the full length of the leucine-binding protein. In one embodiment, the fluorescent protein or its functional fragment or variant thereof of the present invention replaces one or more amino acids between residues 327-331 of the leucine-binding protein or its functional fragment, with the numbering corresponding to the full length of the leucine-binding protein.Preferably, the fluorescent protein or its functional fragment or variant thereof is located between residues 328 / 329 or 329 / 330 of the leucine-binding protein or its functional fragment.

[0069] For example, when the yellow fluorescent protein is inserted between residues 325 / 326, 325 / 327, 325 / 328, 325 / 329, 326 / 327, 326 / 328, 326 / 329, 327 / 328, 327 / 329, or 328 / 329 of the branched-chain amino acid-binding protein shown in SEQ ID NO: 1, the amino acid sequence of the fluorescent probe is as shown in SEQ ID NO: 14~23. For example, when the yellow fluorescent protein is inserted between residues 327 / 328, 327 / 329, 327 / 330, 327 / 331, 328 / 329, 328 / 330, 328 / 331, 329 / 330, 329 / 331, or 330 / 331 of the leucine-binding protein shown in SEQ ID NO: 2, the amino acid sequence of the fluorescent probe is as shown in SEQ ID NO: 24~33.

[0070] In this invention, the responsive polypeptide fused with the fluorophore can be the full length or a fragment of a branched-chain amino acid-binding protein or a leucine-binding protein, preferably amino acids 1-345 of the branched-chain amino acid-binding protein or amino acids 1-347 of the leucine-binding protein.

[0071] "Connector" refers to an amino acid or nucleic acid sequence that links two parts in the polypeptide, protein, or nucleic acid of the present invention. When linking in the polypeptide or protein of the present invention, the length of the connector is no more than 6 amino acids, preferably no more than 4 amino acids, and more preferably 3 amino acids. When linking in the nucleic acid of the present invention, the length of the connector is no more than 18 nucleotides, preferably no more than 12 nucleotides, and more preferably 9 nucleotides.

[0072] When referring to a polypeptide or protein, the term "variant" as used in this invention includes variants that have the same function as the polypeptide or protein but with a different sequence. These variants include (but are not limited to): sequences obtained by deleting, inserting, and / or substituting one or more amino acids (typically 1-30, preferably 1-20, more preferably 1-10, most preferably 1-5) in the sequence of the polypeptide or protein, and sequences obtained by adding one or more amino acids (typically up to 20, preferably up to 10, more preferably up to 5) to its C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the polypeptide or protein. In the art, amino acids of similar properties often refer to amino acid families with similar side chains, which are well-defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, adding one or more amino acids to the C-terminus and / or N-terminus generally does not alter the function of the polypeptide or protein. It is well known to those skilled in the art that in gene cloning, it is often necessary to design suitable restriction enzyme sites, which inevitably introduces one or more irrelevant residues at the end of the expressed polypeptide or protein, without affecting the activity of the target polypeptide or protein. For example, to construct fusion proteins, promote the expression of recombinant proteins, obtain recombinant proteins that are automatically secreted outside host cells, or facilitate the purification of recombinant proteins, it is often necessary to add certain amino acids to the N-terminus, C-terminus, or other suitable regions within the recombinant protein. These additions include, but are not limited to, suitable adaptor peptides, signal peptides, leader peptides, terminal extensions, glutathione S-transferase (GST), maltose E-binding proteins, protein A, tags such as 6His or Flag, or proteolytic sites of factor Xa, thrombin, or enterokinase. Variants of peptides or proteins may include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, peptides or proteins encoded by DNA that can hybridize with the DNA of the peptide or protein under stringent high or low conditions, and peptides or proteins obtained using antiserum against the peptide or protein. These variants may also contain a polypeptide or protein with a sequence identity of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% with respect to the polypeptide or protein.

[0073] In two or more polypeptide or nucleic acid sequences, the term "identity" or "percentage of identity" refers to the similarity of two or more sequences or subsequences, or the similarity of a certain percentage of amino acid residues or nucleotides in a specified region, when compared and matched for maximum correspondence using methods known in the art, such as sequence comparison algorithms, through manual alignment and visual inspection (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%), within a comparison window or specified region. For example, preferred algorithms for determining the percentage of sequence identity and the percentage of sequence similarity are the BLAST and BLAST 2.0 algorithms, see Altschul et al. (1977) Nucleic Acids Res. 25:3389 and Altschul et al. (1990) J. Mol. Biol. 215:403, respectively.

[0074] As used herein, the terms “functional fragment,” “derivative,” and “analyte” refer to a polypeptide or protein that retains substantially the same biological function or activity as the original polypeptide or protein. “Functional fragment,” “derivative,” and “analyte” may have the same or substantially the same amino acid sequence as a naturally occurring protein. “Substantially the same” means that the amino acid sequence is mostly, but not identical, but retains the functional activity associated with its sequence. Typically, two amino acid sequences are “substantially the same” or “substantially homologous” if they are at least 85% identical. Fragments with a three-dimensional structure different from that of a naturally occurring protein are also included. The functional fragments, derivatives, or analogs of the responsive peptides of the present invention may be (i) proteins in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) proteins having substituent groups in one or more amino acid residues; or (iii) proteins formed by fusing a mature protein with another compound (e.g., a compound that extends the protein's half-life, such as polyethylene glycol); or (iv) proteins formed by fusing an additional amino acid sequence to this protein sequence (e.g., a leader sequence or secretion sequence or a sequence used to purify this protein or a proteogenic sequence, or a fusion protein formed with an antigen IgG fragment). Based on the teachings herein, these functional fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0075] The difference between the analogue and the original polypeptide or protein can be a difference in amino acid sequence, a difference in modification that does not affect the sequence, or both. These proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis through radiation or exposure to mutagens, or by site-directed mutagenesis or other known molecular biology techniques.

[0076] The analogues also include analogues having residues different from naturally occurring L-amino acids (such as D-amino acids), and analogues having non-naturally occurring or synthetic amino acids (such as β-, γ-amino acids). It should be understood that the responsive peptides of the present invention are not limited to the representative proteins, fragments, derivatives, and analogues listed above. Modifications (generally without altering the primary structure) include: chemically derived forms of proteins, such as acetylation or carboxylation, either in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications during protein synthesis and processing or further processing steps. This modification can be accomplished by exposing the protein to glycosylation enzymes (such as mammalian glycosylation or deglycosylation enzymes). Modifications also include sequences having phosphorylated amino acid residues (such as phosphotyrosine, phosphotyserine, phosphotythreonine). Proteins modified to improve their resistance to proteolytic hydrolysis or optimize their solubility are also included.

[0077] As used herein, the term "nucleic acid" can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or synthetically produced DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand. The coding region sequence encoding the mature protein can be identical to or a degenerate variant of the coding region sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, or 17. As used herein, "degenerate variant" refers to a nucleic acid sequence encoding the polypeptide of this invention but differing from the coding region sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, or 17.

[0078] When referring to nucleic acids, the term "variant" as used herein can refer to naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include degenerate variants, substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a nucleic acid, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the protein it encodes. The nucleic acids of the present invention may comprise a nucleotide sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity with the nucleic acid sequence.

[0079] As used herein, the term "hybridization under stringent conditions" is used to describe hybridization and washing conditions under which nucleotide sequences typically at least 60% homologous to each other can still hybridize. Preferably, stringent conditions are those under which sequences generally still hybridize to each other with at least 65%, more preferably at least 70%, and even more preferably at least 80% or higher homology. Such stringent conditions are well known to those skilled in the art. A preferred, non-limiting example of stringent conditions is: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 0 °C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42 °C, etc.; or (3) hybridization occurs only when the similarity between the two sequences is at least 90%, more preferably at least 95%. Furthermore, the proteins encoded by the hybridizable nucleic acids have the same biological functions and activities as the mature proteins shown in SEQ ID NO: 4, 5, 6, 7 or 8.

[0080] This invention also relates to nucleic acid fragments that hybridize with the above-described sequences. As used herein, a "nucleic acid fragment" contains at least 15 nucleotides in length, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more. The nucleic acid fragments can be used in nucleic acid amplification techniques (such as PCR).

[0081] The full-length sequence or fragment thereof of the fluorescent probe or fusion protein of this invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed in this invention, especially the open reading frame sequences, and the relevant sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared according to conventional methods known to those skilled in the art. When the sequence is long, two or more PCR amplifications are often required, and then the fragments amplified from each amplification are spliced ​​together in the correct order. When the full-length sequence or fragment thereof of the fluorescent probe or fusion protein is less than, for example, 2500 bp, artificial synthesis can be used. The artificial synthesis method is a conventional DNA synthesis method in the art; for example, the full-length sequence can be obtained by first synthesizing multiple small fragments and then ligating them.

[0082] Recombinant methods can be used to obtain relevant sequences in large quantities. This usually involves cloning the sequence into a vector, transforming it into cells, and then isolating and purifying the relevant polypeptides or proteins from the proliferated host cells using conventional methods.

[0083] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0084] Currently, the DNA sequence encoding the protein of this invention (or its fragments, derivatives, analogs, or variants) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (such as vectors) and cells known in the art. Mutations can be introduced into the protein sequence of this invention through methods such as mutagenic PCR or chemical synthesis.

[0085] The terms "expression vector" and "recombinant vector" used herein are used interchangeably and refer to prokaryotic or eukaryotic vectors well known in the art, such as bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors. These vectors can replicate and remain stable in the host. An important characteristic of these recombinant vectors is that they typically contain expression control sequences. The term "expression control sequence" as used herein refers to an element that can be operatively linked to the target gene to regulate the transcription, translation, and expression of the target gene. This can be an origin of replication, promoter, marker gene, or translation control element, including enhancers, operons, terminators, ribosome binding sites, etc. The choice of expression control sequence depends on the host cell used. Recombinant vectors applicable in this invention include, but are not limited to, bacterial plasmids. In recombinant expression vectors, "operative linking" refers to the linking of the target nucleotide sequence to a regulatory sequence in a manner that allows the expression of the nucleotide sequence. Those skilled in the art are familiar with methods for constructing expression vectors containing the coding sequence of the fusion protein of this invention and suitable transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters include: the *E. coli* lac or trp promoter; the *λ* phage PL promoter; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, retroviral LTRs, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0086] Those skilled in the art will understand that the design of recombinant expression vectors can depend on factors such as the selection of host cells to be transformed and the desired protein expression level. Furthermore, the recombinant expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase or neomycin resistance for eukaryotic cells, or tetracycline or ampicillin resistance for Escherichia coli.

[0087] In one embodiment, the coding sequence of the fluorescent probe or fusion protein of the present invention is digested with BamHI and HindIII and then ligated into a pRSETb vector digested with BamHI and HindIII to obtain an E. coli recombinant expression vector. The expression vector of the present invention can be transferred into host cells to produce a protein or peptide including the fusion protein. This transfer process can be performed using conventional techniques well known to those skilled in the art, such as transformation or transfection.

[0088] In this document, the term "host cell" or "cell," also known as "recipient cell," refers to a cell capable of receiving and containing recombinant DNA molecules. It is the site of recombinant gene amplification. Ideally, a recipient cell should meet the conditions of easy acquisition and proliferation. The "cell" in this invention can include prokaryotic and eukaryotic cells, specifically including bacterial cells, yeast cells, insect cells, and mammalian cells.

[0089] The expression vector of the present invention can be used to express the fluorescent probe or fusion protein of the present invention in prokaryotic or eukaryotic cells. Therefore, the present invention relates to host cells, preferably *E. coli*, into which the expression vector of the present invention has been introduced. The host cell can be any prokaryotic or eukaryotic cell, representative examples including: bacterial cells of *E. coli*, *Streptomyces*, *Salmonella typhimurium*, fungal cells such as yeast, plant cells, insect cells of *Drosophila S2* or *Sf9*, animal cells such as CHO, COS, 293 cells, or Bowes melanoma cells, etc., including but not limited to those host cells mentioned above. The host cell is preferably a variety of cells that are conducive to gene product expression or fermentation production, such cells are well known and commonly used in the art, such as various *E. coli* cells and yeast cells. In one embodiment of the present invention, *E. coli* BL21 is selected to construct the host cell expressing the fusion protein of the present invention. Those skilled in the art will understand how to select appropriate vectors, promoters, enhancers, and host cells.

[0090] As used herein, the terms “transformation” and “transfection,” “conjugation” and “transduction” refer to various techniques known in the art for introducing exogenous nucleic acids (e.g., linear DNA or RNA (e.g., linearized vectors or vectorless standalone gene constructs)) or nucleic acids in vector form (e.g., plasmids, granules, bacteriophages, phage particles, transposons, or other DNA) into host cells, including calcium phosphate or calcium chloride coprecipitation, DEAE-mannan-mediated transfection, lipid transfection, native competent cells, chemically mediated transfer, or electroporation. When the host is a prokaryote such as *Escherichia coli*, DNA-adsorbing competent cells can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. Transformation can also be performed by electroporation if desired. When the host cell is a eukaryotic cell, DNA transfection methods such as calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, and liposome packaging can be used.

[0091] Transformed cells obtained by culture using conventional methods suitable for expression in the host cells can express the fusion protein of the present invention. Depending on the host cells used, the culture medium can be any conventional medium. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature change or chemical induction), and the cells are cultured for a further period.

[0092] The recombinant proteins described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated or purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods. His-tag affinity chromatography is preferred.

[0093] In one embodiment, the fluorescent probe or fusion protein of the present invention is produced by fermentation of Escherichia coli containing the coding sequence of the fusion protein of the present invention, and the pure form of the fluorescent probe or fusion protein of the present invention is obtained by purification by nickel affinity chromatography.

[0094] The uses of the fluorescent probes or fusion proteins of this invention include, but are not limited to: detecting branched-chain amino acids or leucine, detecting branched-chain amino acids or leucine under physiological conditions, detecting branched-chain amino acids or leucine at the subcellular level, and detecting branched-chain amino acids or leucine in situ.

[0095] This invention also provides the application of the fluorescent probes in real-time localization and quantitative detection of branched-chain amino acids (BCAAs) and leucine, as well as in high-throughput compound screening. In one embodiment, the BCAA and leucine fluorescent probes are preferably linked to signal peptides at different sites within the cell and transferred into the cell. Real-time localization of BCAAs and leucine is achieved by detecting the intensity of fluorescence signals in the cell; quantitative detection of the corresponding BCAAs and leucines is performed using standard titration curves. In one embodiment, the standard titration curves for BCAAs and leucine are plotted based on the fluorescence signals of the BCAA and leucine fluorescent probes at different concentrations of BCAAs and leucine. The fluorescent probes of this invention are directly transferred into the cell, eliminating the need for time-consuming sample processing during real-time localization and quantitative detection of BCAAs and leucine, thus improving accuracy. In high-throughput compound screening, the fluorescent probes of this invention add different compounds to the cell culture medium and measure changes in BCAA and leucine content to screen for compounds that affect changes in BCAA and leucine content. Generally, the applications described in this invention do not involve the diagnosis or treatment of diseases.

[0096] In this document, concentrations, contents, percentages, and other values ​​are expressed in range form. It should also be understood that this range form is used for convenience and brevity only, and should be flexibly interpreted to include the values ​​explicitly mentioned at the upper and lower limits of the range, as well as all individual values ​​or subranges included within that range, as if each value and subrange were explicitly mentioned.

[0097] Example

[0098] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0099] The experimental methods described in the following examples, unless otherwise specified, are well known to those skilled in the art and are generally performed under conventional conditions such as those described in Sambrook et al.'s *Molecular Cloning: A Laboratory Manual* (Cold Spring Harbor Laboratory Press, New York, 1989); Jane Rothcams et al.'s *Molecular Biology Laboratory Manual*, translated by J. Sambrook and DW Russell (3rd edition, August 2002, Science Press, Beijing); Fereschney et al.'s *Animal Cell Culture: A Basic Technique Guide* (5th edition), translated by Zhang Jingbo and Xu Cunshuan et al.; J.S. Bonnie Fesnon, M. Dassault et al.'s *A Concise Laboratory Manual of Cell Biology*, translated by Zhang Jingbo et al.; or according to the manufacturer's recommendations. In this document, unless otherwise stated, percentages and parts are by weight. Those skilled in the art can readily implement this invention by making minor modifications and variations to suit specific circumstances, and all such modifications and variations fall within the scope of the claims of this application.

[0100] I. Experimental Materials and Reagents

[0101] The pRSETb-cpYFP, ​​pRSETb-LivJ, and pRSETb-LivK plasmids used in the examples were constructed by the Protein Laboratory of East China University of Science and Technology, and the pRSETb plasmid vector was purchased from Invitrogen. All primers used for PCR were synthesized, purified, and identified correctly by mass spectrometry by Shanghai Jierui Biotechnology Co., Ltd. The expression plasmids constructed in the examples were all sequenced, and the sequencing was performed by BGI Genomics and J. Lee Sequencing. The Taq DNA polymerase used in each example was purchased from Dongsheng Biotechnology, the pfu DNA polymerase from Tiangen Biotech (Beijing) Co., Ltd., and the primeSTAR DNA polymerase from TaKaRa. Corresponding polymerase buffers and dNTPs were included with each purchase. Restriction endonucleases such as BamHI, BglII, HindIII, NdeI, XhoI, EcoRI, and SpeI, as well as T4 ligase and T4 phosphorylase (T4 PNK), were purchased from Fermentas, and corresponding buffers were included with each purchase. The Lip2000 transfection kit was purchased from Invitrogen. Branched-chain amino acids and leucine were purchased from Sigma-Aldrich. Unless otherwise stated, inorganic salts and other chemical reagents were purchased from Sigma-Aldrich. HEPES salt, ampicillin (Amp), and puromycin were purchased from Amersco; the 96-well detection blackboard and the 384-well fluorescence detection blackboard were purchased from Grenier.

[0102] The DNA purification kits used in these examples were purchased from BBI (Canada), and the general plasmid extraction kits were purchased from Tiangen Biotech (Beijing) Co., Ltd. The cloned strain Mach1 was purchased from Invitrogen. Nickel affinity chromatography columns and desalting column packing materials were both from GE Healthcare.

[0103] The main instruments used in the examples are: Biotek Synergy 2 multi-functional microplate reader (Bio-Tek, USA), X-15R high-speed refrigerated centrifuge (Beckman, USA), Microfuge 22R benchtop high-speed refrigerated centrifuge (Beckman, USA), PCR amplifier (Biometra, Germany), ultrasonic disruptor (Ningbo Xinzhi Co., Ltd.), nucleic acid electrophoresis apparatus (Shenneng Bocai Co., Ltd.), fluorescence spectrophotometer (Varian, USA), CO2 constant temperature cell incubator (SANYO), and inverted fluorescence microscope (Nikon, Japan).

[0104] II. Conventional molecular biology methods and cell experimental methods used in the examples

[0105] (a) Polymerase chain reaction (PCR): 1. PCR amplification of the target fragment: This method is mainly used for gene fragment amplification and colony PCR identification of positive clones. The reaction system for PCR amplification is shown in Table 1, and the amplification program is shown in Table 2.

[0106] Table 1. PCR amplification reaction system

[0107] Table 2. PCR Amplification Program 2. Long Fragment (>2500bp) Amplification PCR:

[0108] The long-fragment amplification used in this invention is primarily a reverse PCR amplification vector, a technique used in the following embodiments to obtain site-directed mutagenesis. Reverse PCR primers are designed at the mutation site, with one primer containing the mutated nucleotide sequence at its 5' end. The amplified product then contains the corresponding mutation site. The long-fragment amplification PCR reaction system is shown in Table 3, and the amplification procedure is shown in Table 4 or Table 5.

[0109] Table 3. PCR reaction system for long fragment (>2500 bp) amplification

[0110] Table 4. PCR amplification program for long fragments (>2500bp)

[0111] Table 5. PCR amplification program for long fragments (>2500 bp)

[0112] (II) Endonuclease digestion reaction: The systems for double enzyme digestion of plasmid vectors are shown in Table 6, where n represents the amount of sterile ultrapure water (μL) required to bring the system to its total volume.

[0113] Table 6. Double enzyme digestion system for plasmid vectors

[0114] (iii) Phosphorylation of DNA fragments at the 5' end

[0115] Plasmids or genomes extracted from microorganisms contain phosphate groups at their ends, while PCR products do not. Therefore, a phosphate addition reaction is required at the 5' end of the PCR product. Only DNA molecules with phosphate groups at their ends can undergo ligation. The phosphorylation reaction system is shown in Table 7, where T4 PNK is an abbreviation for T4 polynucleotide kinase, used for the addition reaction of the 5' phosphate group of the DNA molecule.

[0116] Table 7. Phosphorylation reaction system

[0117] (iv) Ligation reaction between target fragment and vector

[0118] The methods for connecting different fragments and carriers vary, and this invention uses three connection methods.

[0119] 1. Blunt-end junctions of blunt-ended short fragments and linearized vectors

[0120] The principle of this method is that the blunt-end product obtained by PCR is phosphorylated at the 5' end of the DNA fragment under the action of T4 PNK, and then ligated with the linearized vector under the action of PEG4000 and T4 DNA ligase to obtain a recombinant plasmid. The homologous recombination ligation system is shown in Table 8.

[0121] Table 8. Homologous recombination linkage system

[0122] 2. Ligation of DNA fragments with sticky ends and vector fragments with sticky ends.

[0123] DNA fragments digested by restriction endonucleases typically produce prominent sticky ends, which can then be ligated to vector fragments containing sequence complementary sticky ends to form recombinant plasmids. The ligation reaction system is shown in Table 9.

[0124] Table 9. Connection Reaction System

[0125] Note: The mass ratio of PCR product fragment to vector double enzyme digestion product is approximately between 2:1 and 6:1.

[0126] 3. Ligation reaction involving the self-circularization of DNA fragment products phosphorylated at the 5' end following site-directed mutagenesis using reverse PCR.

[0127] The 5' phosphorylated DNA fragment was ligated to the 3' and 5' ends of the linearized vector via a self-circularization ligation reaction to obtain a recombinant plasmid. The self-circularization ligation reaction system is shown in Table 10.

[0128] Table 10. Self-cyclization linkage reaction systems

[0129] (V) Preparation and transformation of competent cells

[0130] Preparation of competent cells: 1. Pick a single colony (e.g., Mach1) and inoculate it into 5 mL of LB medium. Incubate overnight at 37 °C on a shaker.

[0131] 2. Take 0.5-1 mL of the overnight culture and transfer it to 50 mL of LB medium. Incubate at 37 ℃ and 220 rpm for 3 to 5 h until the OD600 reaches 0.5.

[0132] 3. Pre-cool the cells in an ice bath for 2 hours.

[0133] Centrifuge at 4.4 ℃ and 4000 rpm for 10 min.

[0134] 5. Discard the supernatant, resuspend the cells in 5 mL of pre-cooled resuspension buffer, and add more resuspension buffer to a final volume of 50 mL after homogenization.

[0135] 6. Ice bath for 45 minutes.

[0136] 7. Centrifuge at 4000 rpm for 10 min at 4 ℃, and resuspend the bacteria in 5 mL of ice-cold storage buffer.

[0137] 8. Place 100 µL of bacterial culture in each EP tube and store at -80 ℃ or in liquid nitrogen.

[0138] Resuspension buffer: CaCl2 (100 mM), MgCl2 (70 mM), NaAc (40 mM)

[0139] Storage buffer: 0.5 mL DMSO, 1.9 mL 80% glycerol, 1 mL 10× CaCl2 (1 M), 1 mL 10× MgCl2 (700 mM), 1 mL 10× NaAc (400 mM), 4.6 mL ddH2O. Conversion: 1. Take 100 μl of competent cells and thaw them on an ice bath.

[0140] 2. Add an appropriate volume of ligation product, gently mix by pipetting, and incubate on ice for 30 minutes. The volume of ligation product added is usually less than 1 / 10 of the competent cell volume.

[0141] 3. Place the bacterial culture in a 42 ℃ water bath for 90 s for heat shock, then quickly transfer it to an ice bath and place it for 5 min.

[0142] 4. Add 500 μl LB and incubate at 200 rpm on a 37 ℃ constant temperature shaker for 1 h.

[0143] 5. Centrifuge the bacterial culture at 4000 rpm for 3 min, and reserving 200 μL of supernatant. Spread the bacterial cells evenly on the surface of an agar plate containing an appropriate amount of antibiotics. Incubate the plate upside down in a 37 ℃ incubator overnight.

[0144] (vi) Protein expression, purification and fluorescence detection

[0145] 1. Transform pRSETb-based branched-chain amino acid and leucine probe plasmids into JM109(DE3), incubate overnight in an upside-down manner, pick clones from the plate into 250 mL Erlenmeyer flasks, place them in a 37 ℃ shaker, and incubate at 220 rpm until OD=0.4~0.8. Add 1 / 1000 (v / v) IPTG (1 M) and induce expression at 18 ℃ for 24~36 h.

[0146] 2. After induction of expression, centrifuge at 4000 rpm for 30 min to collect the bacteria, resuspend the bacterial pellet in 50 mM phosphate buffer, and sonicate until the bacterial cells are clear. Centrifuge at 9600 rpm at 4 ℃ for 20 min.

[0147] 3. The supernatant from centrifugation was purified by a self-assembled nickel affinity chromatography column to obtain the protein. The protein after nickel affinity chromatography was then purified by a self-assembled desalting column to obtain the protein dissolved in 20 mM MOPS buffer (pH 7.4) or phosphate buffered PBS.

[0148] 4. After SDS-PAGE identification of the purified branched-chain amino acids, leucine-binding proteins, and mutant proteins, the probes were diluted to a final concentration of 5–10 μM using assay buffer (100 mM HEPES, 100 mM NaCl, pH 7.3) or phosphate-buffered saline (PBS). Histidine was prepared into a stock solution with a final concentration of 1 M using assay buffer (20 mM MOPS, pH 7.4) or phosphate-buffered saline (PBS).

[0149] Take 100 μl of 5 μM protein solution, incubate at 37 ℃ for 5 min, add branched-chain amino acids and leucine respectively, mix well to a final concentration of 100 mM, and measure the light absorption of the protein at 340 nm using a multifunctional fluorescent microplate reader.

[0150] Take 100 μL of 1 μM fluorescent probe solution, incubate at 37 ℃ for 5 min, add branched-chain amino acids and leucine for titration, and measure the fluorescence intensity emitted at 528 nm after excitation at 485 nm. The fluorescence excitation and emission measurements of the samples were performed using a multifunctional fluorescent microplate reader.

[0151] Take 100 μl of 1 μM fluorescent probe solution, incubate at 37 ℃ for 5 min, add branched-chain amino acids and leucine, and measure the absorption and fluorescence spectra of the probe protein. The absorption and fluorescence spectra of the samples were measured using a spectrophotometer and a fluorescence spectrophotometer.

[0152] (vii) Fluorescence detection in mammalian cells

[0153] 1. The pCDNA3.1+-based branched-chain amino acid and leucine probe plasmid was transfected into HeLa cells using Lipofectamine 2000 (Invitrogen) transfection reagent and cultured in a cell culture incubator at 37 ℃ and 5% CO2. Fluorescence detection was performed 24–36 h after the exogenous gene was fully expressed.

[0154] 2. After the expression was induced, the adherent HeLa cells were washed three times with PBS and placed in HBSS solution for fluorescence microscopy and microplate reader detection.

[0155] Example 1

[0156] Construction of pRSETb-LivJ and pRSETb-LivK plasmids

[0157] The LivJ and LivK genes in *E. coli* were amplified by PCR. The PCR product of LivJ was recovered after gel electrophoresis and digested with BamHI and HindIII. The PCR product of LivK was recovered after gel electrophoresis and digested with EcoRI and HindIII. The pRSETb vector was also double-digested with the corresponding enzymes. Ligation was performed using T4 DNA ligase, and the ligation product was transformed into MachI cells. The transformed MachI cells were plated on LB agar plates (100 μg / mL ampicillin) and incubated overnight at 37 °C. Plasmids were extracted from the grown MachI transformants and identified by PCR. Positive plasmids, after being correctly sequenced, were used for subsequent plasmid construction.

[0158] Construction and detection of plasmids with different insertion sites for pRSETb-LivJ-cpYFP and pRSETb-LivK-cpYFP fluorescent probes.

[0159] In this embodiment, we used pRSETb-LivJ as the base plasmid and selected the plasmids 118 / 119, 118 / 120, 119 / 120, 248 / 249, 248 / 250, 248 / 251, 248 / 252, 248 / 253, 248 / 254, 248 / 255, 248 / 256, 248 / 257, and 248 / 2 based on the LivJ crystal structure. 58, 249 / 250, 249 / 251, 249 / 252, 249 / 253, 249 / 254, 249 / 255, 249 / 256, 249 / 257, 249 / 258, 250 / 251, 250 / 252, 250 / 253, 250 / 254, 250 / 255, 250 / 256, 250 / 257, 250 / 258, 2 51 / 252, 251 / 253, 251 / 254, 251 / 255, 251 / 256, 251 / 257, 251 / 258, 252 / 253, 252 / 254, 252 / 255, 252 / 256, 252 / 257, 252 / 258, 253 / 254, 253 / 255, 253 / 256, 253 / 257, 253 / Positions 258, 254 / 255, 254 / 256, 254 / 257, 254 / 258, 255 / 256, 255 / 257, 255 / 258, 256 / 257, 256 / 258, 257 / 258, 325 / 326, 325 / 327, 325 / 328, 325 / 329, 326 / 327, 326 / 328, 326 / 329, 327 / 328, 327 / 329, and 328 / 329 were detected. Among them, positions 326 / 327 and 327 / 328 showed a response to branched-chain amino acids exceeding 3-fold (e.g., ...). Figure 2 (As shown).

[0160] Based on the pRSETb-LivK base plasmid, plasmids with the following crystal structures were selected according to the LivK crystal structure: 118 / 119, 118 / 120, 119 / 120, 248 / 249, 248 / 250, 248 / 251, 248 / 252, 248 / 253, 248 / 254, 248 / 255, 248 / 256, 248 / 257, 248 / 258, 249 / 250, and 24... 9 / 251, 249 / 252, 249 / 253, 249 / 254, 249 / 255, 249 / 256, 249 / 257, 249 / 258, 250 / 251, 250 / 252, 250 / 253, 250 / 254, 250 / 255, 250 / 256, 250 / 257, 250 / 258, 251 / 252, 251 / 253, 25 1 / 254, 251 / 255, 251 / 256, 251 / 257, 251 / 258, 252 / 253, 252 / 254, 252 / 255, 252 / 256, 252 / 257, 252 / 258, 253 / 254, 253 / 255, 253 / 256, 253 / 257, 253 / 258, 254 / 255, 254 / 256, 25 The following sites were identified: 4 / 257, 254 / 258, 255 / 256, 255 / 257, 255 / 258, 256 / 257, 256 / 258, 257 / 258, 327 / 328, 327 / 329, 327 / 330, 327 / 331, 328 / 329, 328 / 330, 328 / 331, 329 / 330, 329 / 331, and 330 / 331. Among these, sites 328 / 329 and 329 / 330 showed a response to leucine exceeding 1.5-fold (e.g., ...). Figure 2 (As shown).

[0161] DNA fragments of cpYFP were generated using PCR. These fragments were then inactivated by phosphorylation at the 5' end. Simultaneously, linearized vectors pRSETb-LivJ and pRSETb-LivK containing different breakpoints were generated by inverse PCR. The linearized pRSETb-LivJ and pRSETb-LivK were ligated to the 5' phosphorylated cpYFP fragment using PEG4000 and T4 DNA ligase to generate recombinant plasmids. These plates were then used in a Kodak multifunctional in vivo imaging system to select clones exhibiting yellow fluorescence upon FITC channel excitation. Sequencing was performed by the Shanghai branch of Beijing BGI Genomics Co., Ltd.

[0162] After successful sequencing, the recombinant plasmid was transformed into JM109(DE3) to induce expression, and the protein was purified. SDS-PAGE electrophoresis showed a size of 67 kDa. This size is consistent with the size of the His-tag-containing LivJ-cpYFP and LivK-cpYFP fusion proteins expressed by pRSETb-LivJ-cpYFP and pRSETb-LivK-cpYFP. Results are as follows... Figure 1 As shown.

[0163] The purified LivJ-cpYFP and LivK-cpYFP fusion proteins were screened for branched-chain amino acid and leucine responses. The detection signal of the fusion fluorescent protein containing 10 mM branched-chain amino acids and leucine was divided by the detection signal of the fusion fluorescent protein without branched-chain amino acids and leucine.

[0164] The results are as follows Figure 2 As shown, the detection results indicate that the fluorescent probes for branched-chain amino acids (BCAAs) showed a response greater than 3-fold to BCAAs at positions 326 / 327, 327 / 328, 326 / 327, and 327 / 328. The fluorescence intensity at the 528 nm emission site after 420 nm excitation decreased with increasing BCAA concentration, while the fluorescence intensity at the 528 nm emission site after 485 nm excitation at positions 326 / 327 and 327 / 328 increased with increasing BCAA concentration. For leucine, the fluorescent probes showed a response greater than 1.5-fold to leucine at positions 328 / 329, 329 / 330, 328 / 329, and 329 / 330. The fluorescence intensity at the 528 nm emission site after 420 nm excitation decreased with increasing BCAA concentration; while the fluorescence intensity at the 528 nm emission site after 485 nm excitation at positions 328 / 329 and 329 / 330 increased with increasing BCAA concentration.

[0165] Example 2

[0166] Design, construction, and detection of cpBFP-based branched-chain amino acid fluorescent probes

[0167] Following the method described in Example 1, cpYFP was replaced with blue fluorescent protein cpBFP, and fused into branched-chain amino acid-binding protein and leucine-binding protein, respectively, to construct branched-chain amino acid blue fluorescent protein probes and leucine blue fluorescent protein probes. The results are as follows: Figure 3 As shown, the fluorescence detection results indicate that 326 / 327 and 327 / 328 are branched-chain amino acid fluorescent probes that respond to branched-chain amino acids by more than 3 times; and 328 / 329 and 329 / 330 are leucine fluorescent probes that respond to leucine by more than 1.5 times.

[0168] Example 3

[0169] Design, construction, and detection of branched-chain amino acid fluorescent probes based on cpmApple

[0170] Following the method in Example 1, cpYFP was replaced with apple red fluorescent protein cpmApple, and fused into branched-chain amino acid-binding protein and leucine-binding protein to construct branched-chain amino acid red fluorescent protein probes and leucine red fluorescent protein probes, respectively. The results are as follows: Figure 4 As shown, the fluorescence detection results indicate that 326 / 327 and 327 / 328 are branched-chain amino acid fluorescent probes that respond to branched-chain amino acids by more than 3 times; and 328 / 329 and 329 / 330 are leucine fluorescent probes that respond to leucine by more than 1.5 times.

[0171] The results of Examples 1-3 show that the linker region 325-329 of the branched-chain amino acid binding protein is suitable for fusing with cpFP fluorescent protein to obtain a fusion fluorescent protein probe that responds to branched-chain amino acids; the linker region 327-331 of the leucine binding protein is suitable for fusing with cpFP fluorescent protein to obtain a fusion fluorescent protein probe that responds to leucine.

[0172] Example 4

[0173] The simple direct fusion of fluorescent proteins and branched-chain amino acid-binding proteins cannot produce branched-chain amino acid fluorescent probes.

[0174] The fluorescent protein cpYFP was directly fused to the N-terminus or C-terminus of LivK or LivJ proteins to construct branched-chain amino acid fluorescent probes. Then, cpYFP was replaced with blue fluorescent protein cpBFP and apple red fluorescent protein cpmApple to construct branched-chain amino acid blue fluorescent protein probes and red fluorescent protein probes, respectively. The results are as follows: Figure 5 As shown, compared with the control, the fluorescence detection results showed that the above-mentioned branched-chain amino acid fluorescent probes did not change their response to branched-chain amino acids.

[0175] Example 5

[0176] Property detection of LivJ-cpYFP and LivK-cpYFP fluorescent probes

[0177] The purified LivJ-cpYFP and LivK-cpYFP were treated with 0 mM and 5 mM leucine, respectively, for 10 min, and then their fluorescence spectra were detected using a fluorescence spectrophotometer. For excitation spectrum determination: the excitation was fixed at 530 nm, and the excitation spectrum in the 350–515 nm range was detected; for emission spectrum determination: the excitation was fixed at 490 nm, and the emission spectrum in the 505–600 nm range was detected. The spectral curves of the LivJ-cpYFP and LivK-cpYFP fluorescent probes are shown below. Figure 6As shown in A and 6B, the above results indicate that the fluorescence spectral properties of LivJ-cpYFP and LivK-cpYFP fluorescent proteins are similar to those of cpYFP fluorescent protein (Nagai, T. et al., Proc Natl Acad Sci USA. 2001, V.98(6), pp.3197-3202).

[0178] Probes with a detection range of 0.1 µM to 1 mM for branched-chain amino acids, namely F LivJ sensor A (LivJ-cpYFP 326 / 327) and F LivJ sensor B (LivJ-cpYFP 327 / 328), and F LivK sensor A (Livk-cpYFP 328 / 329) and F LivK sensor B (Livk-cpYFP 329 / 330), were selected for the detection of three branched-chain amino acids at concentration gradients (0–1 mM). After treating purified LivJ-cpYFP and LivK-cpYFP for 10 min, the changes in the ratio of fluorescence intensity at 420 nm excitation and 528 nm emission to that at 485 nm excitation and 528 nm emission were measured. The results are as follows: Figure 7 As shown in the image. This allows for the selection of more suitable branched-chain amino acids or leucine for quantitative detection based on their content or level in the sample.

[0179] Example 6

[0180] FLivJ sensor A: Localization of fluorescent probes in different subcellular organelles and detection of fluorescent probe properties within subcellular organelles.

[0181] In this embodiment, we use different localization signal peptides to fuse with FLivJ sensor A, thereby localizing the FLivJ sensor A branched-chain amino acid fluorescent protein probe to different organelles.

[0182] HeLa cells were transfected with plasmids containing the FLivJ sensor A gene fused with different localization signal peptides. After 36 hours, the cells were washed with PBS and then placed in HBSS solution for fluorescence detection using an inverted fluorescence microscope under the FITC channel. We found that FLivJ sensor A, by fusing with different specific localization signal peptides, can localize to various locations including the cytoplasm, nucleus, inner cell membrane, mitochondria, phagosomes, lysosomes, and Golgi apparatus. The results are as follows: Figure 8 As shown, fluorescence was observed in different subcellular structures, and the distribution and intensity of the fluorescence varied.

[0183] HeLa cells were transfected with a plasmid containing the FLivJ sensor A gene, which integrates different localization signal peptides from the cytoplasm and mitochondria. After 36 hours, the cells were washed with PBS and then placed in HBSS solution to detect the changes in the ratio of fluorescence intensity at 420 nm excitation and 528 nm emission to fluorescence intensity at 485 nm excitation and 528 nm emission over a 40-minute period. The results are as follows: Figure 9 The results showed that as branched-chain amino acids were consumed, the ratio 485 / 420 gradually decreased. Then, BCAAs were added, and the test was continued for 43 minutes. The ratio 485 / 420 of the sample with added branched-chain amino acids gradually increased, while the ratio 485 / 420 of the control group continued to decrease until it remained unchanged.

[0184] Example 7

[0185] High-throughput compound screening at the live-cell level based on branched-chain amino acid FLivJ sensor A

[0186] In this embodiment, we used HeLa cells expressing branched-chain amino acid FLivJ sensor A in the cytoplasm for high-throughput compound screening.

[0187] HeLa cells transfected with the FLivJ sensor A gene were washed with PBS, treated with HBSS solution (without branched-chain amino acids) for 1 hour, and then treated with a 10 µM compound for 1 hour. Branched-chain amino acids were then added. The ratio of fluorescence intensity at 420 nm excitation and 528 nm emission to fluorescence intensity at 485 nm excitation and 528 nm emission was recorded using a microplate reader. The sample without any compound treatment was used as the standard. Results are as follows: Figure 10 As shown, we found that most of the compounds used to treat cells had minimal impact on the entry of branched-chain amino acids (BCAAs) into the cells. Sixteen compounds increased the cells' ability to take up BCAAs, while six compounds significantly reduced their uptake.

[0188] Example 8

[0189] The FLivJ sensor A, a fluorescent probe for branched-chain amino acids, is used for the quantification of branched-chain amino acids in different subcellular organelles.

[0190] In this example, we used HeLa cells expressing the branched-chain amino acid FLivJ sensor A in the cytoplasm for semi-quantitative analysis. HeLa cells transfected with the FLivJ sensor A gene were divided into several groups: one group received no treatment; another group was washed with PBS, treated in HBSS solution (without branched-chain amino acids) for 1 hour, then treated with 1 mM of the three branched-chain amino acids for 1 hour; or the cells were left in HBSS solution. The ratio of fluorescence intensity at 420 nm excitation to 528 nm emission and the ratio at 485 nm excitation to 528 nm emission were recorded using a microplate reader. Figure 11 We can detect the metabolism of branched-chain amino acids.

[0191] As can be seen from the above embodiments, the fluorescent probes provided by the present invention have relatively small protein molecular weights and are easy to mature, exhibit large fluorescence dynamic changes, good specificity, and can be expressed in cells through gene manipulation methods. They can be used to locate and quantify branched-chain amino acids in and out of cells in real time, and can also be used for high-throughput compound screening.

[0192] Other implementation methods

[0193] This specification describes many embodiments. However, it should be understood that those skilled in the art can make various modifications and refinements without departing from the principles of the invention, and these modifications and refinements should also be considered within the scope of protection of this invention.

Claims

1. A fluorescent probe comprising a) Peptides that respond to leucine, and b) Optically active peptides, wherein The leucine-responsive polypeptide is shown in SEQ ID NO: 1 or 2 or a functional fragment thereof. The optically active polypeptide is shown in any of SEQ ID NO: 3-13. The optically active peptide is located at any of the following sites in the leucine-responsive peptide shown in SEQ ID NO: 1: 118 / 119, 118 / 120, 119 / 120, 248 / 252, 248 / 254, 249 / 250, 249 / 255, 249 / 257, 249 / 258, 250 / 258, 251 / 252, 251 / 254, 251 / 255, 251 / 256, 253 / 254, 253 / 258, 254 / 255, 254 / 256, 254 / 257, 255 / 256, 255 / 257, 325 / 326, 325 / 327, 325 / 328, 325 / 329, 326 / 327, 326 / 328, 326 / 329, 327 / 328, 327 / 329, 328 / 329, 248 / 255, 248 / 256, 251 / 257, 251 / 258, 252 / 254, 252 / 256 or 256 / 257, or The optically active polypeptide is located at residues of the leucine-responsive polypeptide shown in SEQ ID NO: 2 selected from one or more of the following sites: 249 / 255, 249 / 257, 250 / 258, 251 / 256, 253 / 254, 254 / 257, 328 / 329, 328 / 330, 329 / 330, or 252 / 256. Preferably, The optically active polypeptide is located at residues of the leucine-responsive polypeptide shown in SEQ ID NO: 1 selected from one or more of the following sites: 251 / 255, 325 / 326, 325 / 327, 325 / 328, 325 / 329, 326 / 327, 326 / 328, 326 / 329, 327 / 328, or The optically active polypeptide located at residues of the leucine-responsive polypeptide shown in SEQ ID NO: 2 is selected from one or more of the following sites: 328 / 329, 328 / 330, 329 / 330.

2. The fluorescent probe of claim 1, wherein the leucine-responsive polypeptide also responds to other branched-chain amino acids, preferably, the other branched-chain amino acids are isoleucine or valine.

3. The fluorescent probe of any one of claims 1-2, further comprising one or more linkers or localization sequences between the leucine-responsive polypeptide and the optically active polypeptide.

4. The nucleic acid sequence or its complementary sequence encoding the fluorescent probe of any one of claims 1-3.

5. An expression vector comprising the nucleic acid sequence of claim 4, which is operatively linked to an expression control sequence.

6. Cells comprising the expression vector of claim 5.

7. A method of preparing the fluorescent probe according to any one of claims 1 to 3, comprising: Provide a cell comprising a vector expressing the fluorescent probe of any one of claims 1-3, culture the cell under conditions of cell expression, and isolate the fluorescent probe.

8. A method of detecting branched chain amino acids comprising: The fluorescent probe as described in any one of claims 1-3 or the fluorescent probe prepared by the method described in claim 7 is brought into contact with a sample, and changes in optically active peptides are detected, wherein the branched-chain amino acids are selected from one or more of leucine, isoleucine and valine.

9. Use of the fluorescent probe according to any one of claims 1-3 or the fluorescent probe prepared by the method according to claim 7 in detecting branched-chain amino acids in a sample, wherein the branched-chain amino acids are selected from one or more of leucine, isoleucine and valine.

10. A kit comprising a fluorescent probe according to any one of claims 1-3 or a fluorescent probe prepared by the method of claim 7.

Images