WO2018187942A1 - Procaryotic protein for screening binder for glucose transporter glut and preparation method and use thereof - Google Patents

Abstract

Disclosed is a prokaryotic homologous protein of GLUT, a method for preparing the prokaryotic homologous protein, a method for screening a binder for GLUT using the prokaryotic homologous protein, and a kit comprising the prokaryotic homologous protein for screening the binder for GLUT.

Description

Prokaryotic protein for screening for binding agent for glucose transporter GLUT, preparation method and use thereof Technical field

The present disclosure relates to prokaryotic proteins for screening for binding agents to the glucose transporter GLUT, and methods for their preparation and use. More specifically, the present disclosure relates to a prokaryotic homologous protein of GLUT which can be used in drug development, a method for producing the prokaryotic homologous protein, a method for screening a binding agent against GLUT using the prokaryotic homologous protein, and a prokaryotic core comprising the same A kit of homologous proteins for screening for binding agents to GLUT.

Background technique

GLUT is an important protein that mediates the transport of glucose across the membrane. In addition to the main function of transporting glucose molecules, different types of GLUT can also transport monosaccharides such as galactose, mannose, fructose, and xylose. Further, part of the GLUT can also transport non-saccharide substances such as inositol, uric acid, glucosamine, etc. (Non-Patent Document 1).

The GLUT is encoded by the SLC2 gene and consists of approximately 500 amino acids. Fourteen GLUT proteins have been identified in the human genome, all of which are members of the Major facilitator superfamily (MFS). Members of GLUT have a typical MFS structure, and their primary amino acid sequence is folded to form 12 transmembrane helix (TM), in which the first 6 transmembrane helices form the N-terminal domain, and the last 6 transmembrane helices form C. In the terminal domain, two transmembrane domains are linked by a soluble domain in the middle of the N/C-terminal transmembrane domain (Non-Patent Document 2). As a representative of the members of GLUT, the nucleotide sequence and amino acid sequence of GLUT1 are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

The process of transporting substrates by GLUT can be described by alternate open models (Non-Patent Document 3). The substrate binding site of GLUT is located in the middle of the N/C terminal domain, and some amino acid residues on the N-terminal domain and the C-terminal domain contribute to the binding of the substrate. In the direction perpendicular to the cell membrane, the binding site of the substrate is located approximately in the middle of the cell membrane. During the transmembrane transport of the substrate, the N/C-terminal domain of GLUT opens to the side of the cell membrane, and the hydrophilic channel formed by the substrate enters the substrate binding site located in the center of the cell membrane. . The N/C-terminal domain then opens the substrate binding site to the other side of the cell membrane by conformational changes. The substrate is released to the other side of the cell membrane by a new hydrophilic channel formed by the other side opening. Thereafter, the N/C terminal domain undergoes a conformational change again, idling back to the initial state without carrying the substrate, thereby completing the transmembrane transport process of the entire substrate (Non-Patent Documents 4 and 5). Fig. 1 schematically shows the above process (Non-Patent Document 6).

Due to the important role of GLUT in the process of cellular glucose transport, inactivating mutations or expression disorders of GLUT are associated with numerous diseases such as GLUT1 deficiency syndrome, Fanconi-Bickel syndrome, type 2 diabetes, and the like (Non-Patent Document 1). In addition, overexpression of GLUT has been identified in many cancer cells, such as lymphoma, colorectal cancer, hepatocellular carcinoma, head and neck cancer, stomach cancer, prostate cancer, thyroid cancer, kidney cancer, lung cancer, pancreatic cancer, sarcoma. , laryngeal cancer, esophageal cancer, brain cancer, breast cancer, choriocarcinoma, ovarian cancer, endometrial cancer, retinoblastoma, rhabdomyosarcoma, glioma, cervical cancer, gallbladder cancer, oral cancer, squamous cell carcinoma Bladder cancer, multiple myeloma, melanoma, testicular seminoma, etc. (Non-Patent Document 7, Non-Patent Document 15). Because cancer cells have altered metabolic pathways, they need to provide energy to cells through glycolysis, an inefficient method of ATP synthesis. This phenomenon is known as the Warburg effect. Therefore, cancer cells need to express more glucose transporters to enhance glucose absorption, thus ensuring the energy supply of cells. At present, treatment methods for designing GLUT inhibitors to suppress and kill cancer cells have been paid more and more attention, and studies have shown that the addition of GLUT inhibitors to cancer cells can inhibit the growth of cancer cells (Non-Patent Literature) 8, Non-Patent Document 15).

In drug development efforts to design GLUT inhibitors for the Weinberg effect, the current primary screening method is based on cell or liposome substrate transport inhibition assays. Although protein-binding agent binding assays are a more direct and efficient screening method, there is currently no method for efficiently determining the affinity of GLUT proteins for small molecules due to the limitations of GLUT's own stability. Therefore, there is an urgent need for a stable protein model capable of simulating GLUT.

The prokaryotic homologous protein of GLUT is known as follows: GlcPse protein [Staphylococcus epidermidis] (SEQ ID NO: 52), xylose transporter XylE [Escherichia coli] (SEQ ID NO: 4), D-glucose -Proton symporter [Bifidobacterium] Adolescentis) ATCC 15703] (GenBank: BAF40314.1), Bifidobacterium merycicum (GenBank: KFI68887.1), Glucose/mannose: H+ symporter GlcP [Brevibacterium linens] (GenBank: AOP54074.1), glucose/mannose: H+ symporter GlcP [Frigoribacterium sp. JB110] (GenBank: SJM63274.1), glucose/mannose: H+ symporter GlcP [Arthrobacter rhombi] (GenBank :SJM45571.1), MFS transporter [Lactobacillus spicheri] (GenBank: WP_045806284.1), MFS transporter [Lactobacillus hammesii] (GenBank: WP_057734814.1), MFS transporter-sugar carrier (SP) family [Flavobacterium phragmitis] (GenBank: SFD98248.1), MFS transporter [Bacteroidales bacterium 43_8] (GenBank: OKZ35062.1), MFS transporter [Corynebacterium stationis] (GenBank: WP_066796074.1) and the like. Since prokaryotic proteins are generally easier to express and purify by molecular biological techniques than their eukaryotic homologs, and generally have better stability in solution, consider prokaryotic homologous proteins using GLUT when screening for binding agents to GLUT. To simulate GLUT.

Among them, the GlcPse protein is derived from Staphylococcus epidermidis, which has a sequence similarity with GLUT of up to 49% to 58%. The resolved three-dimensional structural information indicates that the GlcPse protein has the same MFS structure as GLUT, the amino acid residues of its substrate binding site are highly conserved compared to GLUT, and it also transports the substrate through an alternate open model. Further transport experiments have also shown that GlcPse is capable of specifically transporting glucose, and this transport activity can be inhibited by a partial GLUT inhibitor (Non-Patent Document 10).

The xylose transporter XylE is derived from Escherichia coli and has a sequence similarity of 47% to 51% with GLUT. The inventors first analyzed the three-dimensional structure of the XylE protein in the world (Non-Patent Document 9). By analyzing the structural information, XylE has the same MFS structure as GLUT, its substrate binding site amino acid residues are highly conserved with GLUT, and XylE also transports substrates through alternate open models.

A major difficulty in the design of drugs for transporters is how to distinguish the different conformations of transporters. In different conformations (such as the inward opening and the outward opening), the spatial position of the amino acid residues associated with substrate binding may change, resulting in different binding properties of the substrate, the drug molecule and the transporter ( Non-patent documents 11 and 12). Without distinction, it is difficult to rationally design potential drug molecules in further drug design optimization.

However, obtaining a stable transporter conformation is itself one of the difficulties in the field of transporter research, and no stable conformational conformation of GLUT has been reported. At present, in the drug design process for transporters, only the stage of computer-simulated drug-assisted drug design can realize drug design for transporters with specific conformations, but there is no effective differentiation method in the stage of screening drug molecules.

A method of immobilizing LacY to an outward-opening conformation by introducing a mutation of a tryptophan residue into an Escherichia coli-derived lactose transporter LacY is known (Non-Patent Document 13). However, LacY is neither capable of transporting nor binding to the substrate glucose of GLUT (Non-Patent Document 14), and thus cannot be used as a prokaryotic protein model of GLUT. There is an urgent need in the art for prokaryotic protein models capable of mimicking the conformational isomer of GLUT, as well as methods for preparing such prokaryotic protein models. In addition, there is a need to establish an efficient method for screening binding agents for GLUTs using prokaryotic protein models of GLUT.

Prior art literature

Non-patent literature

Non-Patent Document 1: Mueckler, M. & Thorens, B. The SLC 2 (GLUT) family of membrane transporters. Molecular aspects of medicine 34, 121-138, doi: 10.1016/j.mam. 2012.07.001 (2013).

Non-Patent Document 2: Deng, D. & Yan, N. GLUT, SGLT, and SWEET: Structural and mechanistic investigations of the glucose transporters. Protein science: a publication of the Protein Society 25, 546-558, doi: 10.1002/pro .2858 (2016).

Non-Patent Document 3: Jardeczky, O. Simple allosteric model for membrane pumps. Nature 211, 969-970 (1966).

Non-Patent Document 4: Deng, D. et al. Crystal structure of the human glucose transporter GLUT1. Nature 510, 121-125, doi: 10.1038/nature 13306 (2014).

Non-Patent Document 5: Deng, D. et al. Molecular basis of of ligand recognition and transport by glucose transporters. Nature 526, 391-396, doi: 10.1038/nature14655 (2015).

Non-Patent Document 6: Yan, N. Structural advances for the major facilitator superfamily (MFS) transporters. Trends in biochemical sciences 38, 151-159, doi: 10.1016/j.tibs. 2013.01.003 (2013).

Non-Patent Document 7: Szablewski, L. Expression of glucose transporters in cancers Biochimica et biophysica acta 1835, 164-169, doi: 10.1016/j.bbcan. 2012.12.004 (2013).

Non-Patent Document 8: Zhao, Y., Butler, E. B. & Tan, M. Targeting cellular metabolism to improve cancer therapeutics. Cell death & disease 4, e532, doi: 10.1038/cddis. 2013. 60 (2013).

Non-Patent Document 9: Sun, L. et al. Crystal structure of a journal homologue of glucose transporters GLUT 1-4. Nature 490, 361-366, doi: 10.1038/nature11524 (2012).

Non-Patent Document 10: Iancu, CV, Zamoon, J., Woo, SB, Aleshin, A. & Choe, JYCrystal structure of a glucose/H+symporter and its mechanism of action. Proceedings of the National Academy of Sciences of the United States of America 110, 17862-17867, doi: 10.1073/pnas.1311485110 (2013).

Non-Patent Document 11: Quistgaard, EM, Low, C., Moberg, P., Tresaugues, L. & Nordlund, P. Structural basis for substrate transport in the GLUT-homology family of monosaccharide transporters. Nature structural & molecular biology 20, 766-768, doi: 10.1038/nsmb.2569 (2013).

Non-Patent Document 12: Wisedchaisri, G., Park, MS, Iadanza, MG, Zheng, H. & Gonen, T. Proton-coupled sugar transport in the prototypical major facilitator superfamily protein XylE. Nature communications 5, 4521, doi: 10.1038 /ncomms5521(2014).

Non-Patent Document 13: Irina Smirnova, Vladimir Kasho, Junichi Sugihara, and H. Ronald Kaback. Trp replacements for tightly interacting Gly-Gly pairs in LacY stabilize an outward-facing conformation. Proceedings of the National Academy of Sciences of the United States of America 110,8876-8881, doi:10.1073/pnas.1306849110 (2013).

Non-Patent Document 14: Hemant Kumar, Vladimir Kasho, Irina Smirnova, Janet S. Finer-Moorea, H. Ronald Kaback, and Robert M. Stroud. Structure of sugar-bound LacY. Proceedings of the National Academy of Sciences of the United States Of America 111,1784-1788, doi:10.1073/pnas.1324141111 (2014).

Non-Patent Document 15: Alison N. McCracken, Aimee L. Edinger. Nutrient transporters: the Achilles' heel of anabolism. Trends in Endocrinology & Metabolism. Volume 24, Issue 4, p200-208, April 2013.

Summary of the invention

technical problem

In view of this, the technical problem to be solved by the present disclosure is to provide a prokaryotic protein model for determining the binding of GLUT to a candidate molecule. Preferably, the prokaryotic protein model can mimic a specific conformer of the GLUT.

Further, the technical problem to be solved by the present disclosure is to provide a method of preparing and producing a prokaryotic protein model for determining the binding of a GLUT to a candidate molecule. Preferably, the prokaryotic protein model can mimic a specific conformer of the GLUT.

In addition, the technical problem to be solved by the present disclosure is to provide a method for determining the binding of GLUT to a candidate molecule by using a prokaryotic homologous protein of GLUT as a model, and using a prokaryotic homologous protein of GLUT as a model to screen and identify against GLUT. The method of binding agent.

In addition, the technical problem to be solved by the present disclosure is to provide a kit for screening and identifying a binding agent for GLUT, the kit comprising a prokaryotic protein model of GLUT.

In addition, the technical problem to be solved by the present disclosure is to provide a method of immobilizing the conformation of prokaryotic homologous proteins of GLUT. Preferably, the prokaryotic protein to which the conformation is immobilized can mimic a specific conformer of the GLUT.

solution

In order to solve the above problems, the inventors have conducted intensive studies and found that a prokaryotic homologous protein of GLUT is used as a prokaryotic protein model of GLUT, and further, by using a large sterically hindered side chain amino acid residue on a prokaryotic homologous protein of GLUT A base-mutated prokaryotic protein that efficiently screens for binding agents (eg, drug molecules) to GLUT. Among them, the prokaryotic homologous protein with a large sterically hindered side chain amino acid residue mutation can be used as a prokaryotic protein model of the outward-opening GLUT conformer.

Further, the inventors have found a method of preparing and producing a prokaryotic protein model of the above GLUT. And a method of immobilizing the conformation of the prokaryotic homologous protein by introducing a large sterically hindered side chain amino acid residue mutation into a prokaryotic homologous protein of GLUT, wherein the prokaryotic homologous protein can be immobilized as an outward open conformational isomer.

In a possible embodiment of the first aspect of the present disclosure, there is provided a method of screening a binding agent for a GLUT, the method comprising the steps of 1) and/or 2) below, and the step comprising 3):

1) a step of detecting binding of a candidate molecule to a first prokaryotic protein,

2) Steps of detecting the binding of the candidate molecule to the second prokaryotic protein

3) determining the binding of the candidate molecule to the GLUT, thereby determining whether the candidate molecule is a binding agent for the GLUT;

Wherein the first prokaryotic protein is a prokaryotic-derived homologous protein of GLUT, the first prokaryotic protein capable of binding to glucose, the GLUT being selected from the group consisting of GLUT1, GLUT2, GLUT3, GLUT4, GLUT5, GLUT6, GLUT7, GLUT8, GLUT9 a group consisting of GLUT10, GLUT11, GLUT12, GLUT13, GLUT14, and other GLUT subtypes, and the first prokaryotic protein has more than 35% sequence similarity to the GLUT;

The second prokaryotic protein is a protein comprising a sequence having a large sterically hindered side chain amino acid residue mutation in an amino acid sequence of the first prokaryotic protein, which mimics a conformational isomer of GLUT;

The large sterically hindered side chain amino acid is selected from the group consisting of tryptophan, tyrosine, phenylalanine, lysine, arginine, glutamic acid, glutamine, aspartic acid, and asparagine. group.

In another possible embodiment of the first aspect of the present disclosure, in step 3), the combination of the candidate molecule and the GLUT is determined as follows: a), b) and/or c):

a) if the binding of the candidate molecule to the first prokaryotic protein is detected in step 1), it is determined that the candidate molecule is capable of binding to the GLUT;

b) if the binding of the candidate molecule to the second prokaryotic protein is detected in step 2), determining that the candidate molecule is capable of binding to the conformational isomer of the GLUT mimicked by the second prokaryotic protein;

c) if the binding of the candidate molecule to the first prokaryotic protein is detected in step 1), and the binding of the candidate molecule to the second prokaryotic protein is not detected in step 2), then the candidate molecule is determined to be different from said The conformational isomer of the GLUT of the conformational isomer of the two prokaryotic proteins.

In another possible embodiment of the first aspect of the present disclosure, the second prokaryotic protein mimics an outward open-ended conformer of the GLUT.

In another possible embodiment of the first aspect of the present disclosure, in step 3), the combination of the candidate molecule and the GLUT is determined as follows: a), b) and/or c):

a) if the binding of the candidate molecule to the first prokaryotic protein is detected in step 1), it is determined that the candidate molecule is capable of binding to the GLUT;

b) if the binding of the candidate molecule to the second prokaryotic protein is detected in step 2), determining that the candidate molecule is capable of binding to the outward open conformational conformation of the GLUT;

c) if the binding of the candidate molecule to the first prokaryotic protein is detected in step 1), and the binding of the candidate molecule to the second prokaryotic protein is not detected in step 2), then the candidate molecule can be determined to be inward-open with the GLUT Conformational isomers are combined.

In another possible embodiment of the first aspect of the present disclosure, the detecting is performed using a micro thermophoresis method and/or an isothermal calorimetric method.

In a possible embodiment of the second aspect of the present disclosure, a kit for screening a binding agent against a GLUT, the kit comprising a first prokaryotic protein and a second prokaryotic protein;

Wherein the first prokaryotic protein is a prokaryotic-derived homologous protein of GLUT, the first prokaryotic protein capable of binding to glucose, the GLUT being selected from the group consisting of GLUT1, GLUT2, GLUT3, GLUT4, GLUT5, GLUT6, GLUT7, GLUT8, GLUT9 a group consisting of GLUT10, GLUT11, GLUT12, GLUT13, GLUT14, and other GLUT subtypes, and the first prokaryotic protein has more than 35% sequence similarity to the GLUT;

The second prokaryotic protein is a protein comprising a sequence having a large sterically hindered side chain amino acid residue mutation in an amino acid sequence of the first prokaryotic protein, which mimics a conformational isomer of GLUT;

The large sterically hindered side chain amino acid is selected from the group consisting of tryptophan, tyrosine, phenylalanine, lysine, arginine, glutamic acid, glutamine, aspartic acid, and asparagine. group.

In another possible embodiment of the second aspect of the present disclosure, the second prokaryotic protein mimics an outward open-ended conformer of the GLUT.

In a possible embodiment of the third aspect of the present disclosure, there is provided an isolated prokaryotic protein mimicking a GLUT, which is a prokaryotic-derived homologous protein of GLUT, wherein the prokaryotic protein is capable of binding glucose,

The GLUT is selected from the group consisting of GLUT1, GLUT2, GLUT3, GLUT4, GLUT5, GLUT6, GLUT7, GLUT8, GLUT9, GLUT10, GLUT11, GLUT12, GLUT13, GLUT14, and other GLUT subtypes, and the prokaryotic protein and the GLUT Has a sequence similarity of more than 35%.

In another possible embodiment of the third aspect of the present disclosure, there is provided an isolated prokaryotic protein which mimics the conformational isomer of GLUT, which is an amino acid sequence of a homologous protein derived from a prokaryote derived from GLUT a protein having a sequence of a large sterically hindered side chain amino acid residue mutated by a prokaryotic-derived homologous protein of the GLUT,

Wherein the GLUT is selected from the group consisting of GLUT1, GLUT2, GLUT3, GLUT4, GLUT5, GLUT6, GLUT7, GLUT8, GLUT9, GLUT10, GLUT11, GLUT12, GLUT13, GLUT14, and other GLUT subtypes, and the prokaryotic source of the GLUT a homologous protein having more than 35% sequence similarity to the GLUT, and

The large sterically hindered side chain amino acid is selected from the group consisting of tryptophan, tyrosine, phenylalanine, lysine, arginine, glutamic acid, glutamine, aspartic acid, and asparagine. group.

In another possible embodiment of the third aspect of the present disclosure, the prokaryotic protein mimics an outward open-ended conformer of GLUT.

In a possible embodiment of the fourth aspect of the present disclosure, a polynucleotide encoding a prokaryotic protein provided by the third aspect of the present disclosure is provided.

In another possible embodiment of the fourth aspect of the present disclosure, an expression vector comprising the above polynucleotide is provided.

In another possible embodiment of the fourth aspect of the present disclosure, there is provided a transformant obtained by transforming a host with the above expression vector, preferably Escherichia coli.

In another possible embodiment of the fourth aspect of the present disclosure, a method for producing a prokaryotic protein simulating a conformational isomer of GLUT or GLUT, which is prepared by using the above polynucleotide, expression vector or transformant, is provided. The prokaryotic protein.

In a possible embodiment of the fifth aspect of the present disclosure, there is provided a method of immobilizing a conformation of a prokaryotic-derived homologous protein of GLUT, characterized by a homologue derived from a prokaryote of said GLUT The protein introduces a large sterically hindered side chain amino acid residue mutation to fix the same A conformation of a source protein, wherein the homologous protein is capable of binding to glucose, the GLUT being selected from the group consisting of GLUT1, GLUT2, GLUT3, GLUT4, GLUT5, GLUT6, GLUT7, GLUT8, GLUT9, GLUT10, GLUT11, GLUT12, GLUT13, GLUT14, and other GLUTs a group consisting of subtypes, and the homologous protein has more than 35% sequence similarity to the GLUT, and

The large sterically hindered side chain amino acid is selected from the group consisting of tryptophan, tyrosine, phenylalanine, lysine, arginine, glutamic acid, glutamine, aspartic acid, and asparagine. group.

In another possible embodiment of the fifth aspect of the present disclosure, the homologous protein is immobilized in an outwardly open conformation.

Beneficial effect

The present disclosure provides prokaryotic protein models of the glucose transporter GLUT, which can serve as a tool protein for efficient screening of binding agents (eg, drug molecules) to GLUTs. Based on these prokaryotic proteins, the present disclosure also provides methods for efficiently screening for binding agents (eg, drug molecules) to GLUTs, as well as kits for screening for binding agents to GLUTs. Compared to previous screening methods, the disclosed method not only provides direct binding information of the protein to the candidate molecule, but also distinguishes the positional information of the binding agent binding to the GLUT. This important information can, for example, enable researchers to further optimize the potential drug molecules that have been initially screened, thereby accelerating drug development efforts.

Further features and aspects of the present disclosure will become apparent from the following detailed description of exemplary embodiments.

DRAWINGS

The accompanying drawings, which are incorporated in FIG

Figure 1 is a schematic representation of a substrate transport model for GLUT proteins. Wherein N represents an N-terminal domain and C represents a C-terminal domain.

Figure 2 is a graph showing the alignment of amino acid sequences of XylE with GLUT1, GLUT2, GLUT3 and GLUT4. The site marked by "*" in Figure 2 corresponds to a conserved site in the amino acid sequence of XylE.

Figure 3 is a graph showing the results of purification of XylE protein. Wherein a in Fig. 3 is a graph showing the purification results of the XylE protein obtained by size exclusion chromatography, and b in Fig. 3 is a graph showing the results of SDS polyacrylamide gel electrophoresis (SDS-PAGE) of the purified XylE protein.

Figure 4 is a graph showing the results of purification of the XylE-X0 mutant protein. Wherein a in FIG. 4 is a map showing the purification result of the XylE-X0 mutant protein by size exclusion chromatography, and b in FIG. 4 is a SDS-PAGE result diagram of the purified XylE-X0 mutant protein.

Figure 5 is a graph showing the atomic-scale resolution structure of the XylE-X0 mutant protein. Mutations in the two large sterically hindered side chain amino acid residues of the XylE-X0 mutant are indicated by G58W and L351W, respectively. TM2 stands for "second transmembrane helix", and other transmembrane helices have the same shorthand way.

Figure 6 is a graph showing the structural alignment of the XylE-X0 mutant and the wild-type XylE protein at the substrate binding site.

Figure 7 is a graph showing the results of liposome transport experiments of XylE-X0 mutants or wild-type XylE proteins. A in Figure 7 shows the results of a substrate (3H isotope-labeled xylose) transport experiment using liposomes carrying wild-type XylE protein or XylE-X0 mutant, respectively. b in Fig. 7 is a graph showing the results of SDS-PAGE showing the amount of protein inserted in the liposome, wherein "M" represents a molecular weight marker (Marker), and "control" represents a liposome negative control not loaded with a protein, "WT" is the result of liposome carrying the wild-type XylE protein, and "G58W/L315W" is the result of liposome carrying the XylE-X0 mutant.

Figure 8 is a graph showing the results of a polyethylene glycol labeling experiment. Wherein a in Figure 8 is a schematic representation of a polyethylene glycol label (mPEG-Mal-5K) for a Cys-less mutant in wild-type XylE (denoted as "No Cys" a marker pathway for the marker-derived mutant (referred to as "I171C & WT") introduced by single point mutation of Ile171Cys, and b in Figure 8 schematically represents mPEG-Mal-5K for the "Cys-free" mutant A labeling pathway for a marker mutant (referred to as "I171C & G58W/L315W") in which the Ile171Cys mutation and the Gly58Trp and Leu315Trp mutations were introduced was introduced. c in Fig. 8 is a graph showing the results of Western Blot of each mutant protein after the cross-linking experiment, showing that the ultrasonic destruction is performed without or without In the case of cells, the labeling results of mPEG-Mal-5K for the "no Cys" mutant, the "I171C & WT" mutant, and the "I171C & G58W/L315W" mutant.

Figure 9 is a graph showing the binding of wild-type XylE protein ("XylE WT") or XylE-X0 mutant ("XylE-X0") to xylose or glucose as determined by isothermal calorimetry. "μM" in the figure indicates μmol/L.

Figure 10 is a graph showing the binding of wild-type XylE protein ("XylE WT") or XylE-X0 mutant ("XylE-X0") to xylose or glucose as determined by microcalorimetry. "μM" in the figure indicates μmol/L.

Figure 11 is a graph showing the inhibitory effects of two GLUT inhibitors on the transport activity of XylE. The transport activity inhibitory effect was determined by a proteoliposome transport assay. "Control" indicates the result of an empty liposome negative control to which no protein was added, "WT" indicates the result of transport activity of wild-type XylE protein when not added with a GLUT inhibitor (set to 100%), "CCB" and "rootsin" The results of the transport activity of the wild-type XylE protein when cytochalasin B (CCB) and phloretin were added, respectively.

Figure 12 is a graph showing the binding of phloretin or CCB to wild-type XylE ("XylE WT") or XylE-X0 mutant ("XylE-X0") as determined by microcalorimetry. "μM" in the figure indicates μmol/L.

Figure 13 is a graph showing the results of purification of GlcPse protein. Wherein a in Fig. 13 is a graph showing the result of purification of the GlcPse protein obtained by size exclusion chromatography, and b in Fig. 13 is a graph showing the results of SDS-PAGE of the purified GlcPse protein.

Figure 14 is a graph showing the results of purification of a GlcP-6 mutant protein. Wherein a in Fig. 14 is a graph showing the result of purification of the GlcP-6 mutant obtained by size exclusion chromatography, and b in Fig. 14 is a graph showing the results of SDS-PAGE of the purified GlcP-6 mutant.

Figure 15 is a graph showing the binding of wild-type GlcPse protein ("GlcPse") or GlcP-6 mutant ("GlcP-6") to glucose as determined by isothermal calorimetry.

Figure 16 is a graph showing the binding of wild-type GlcPse protein ("GlcPse") or GlcP-6 mutant ("GlcP-6") to glucose as determined by microcalorimetry.

17A-17C are graphs showing the binding of phloretin to mutant proteins of XylE-X1 to XylE-X23 as determined by microcalorimetry. Here, "X1 to X23" represents the result of binding of phloretin to a mutant of XylE-X1 to XylE-X23, and the same applies hereinafter. "μM" in the figure indicates μmol/L.

Figure 18 is a graph showing the results of screening a small molecule compound test library using wild-type XylE protein ("XylE WT") and XylE-X0 mutant ("XylE-X0"). Among them, a in Fig. 18 shows a positive screening result, and Phloridzin and Fasentin which are bound to both the wild type XylE protein and the XylE-X0 mutant are selected. b in Figure 18 shows the negative screening results, and D-ribose does not bind to either the wild-type XylE protein or the XylE-X0 mutant. The binding of each small molecule compound in the library to the XylE or XylE-X0 mutant was determined by microcalorimetry. "μM" in the figure indicates μmol/L.

Fig. 19 is a graph showing the results of an experiment for inhibiting the transport activity of phlorizin and farestein to XylE. The transport activity inhibitory effect was determined by a proteoliposome transport assay. "Control" indicates the result of an empty liposome negative control to which no protein was added, and "WT" indicates the result of transport activity of XylE when no phlorizin or farestein was added (set to 100%), "root glucoside" and " "Farsentin" indicates the results of the transport activity of XylE when phlorizin and farestein were added, respectively.

detailed description

Various exemplary embodiments, features, and aspects of the present disclosure are described in detail below with reference to the drawings. The word "exemplary" is used exclusively herein to mean "serving as an example, embodiment, or illustrative." Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or preferred.

In addition, numerous specific details are set forth in the Detailed Description of the <RTIgt; Those skilled in the art will appreciate that the present disclosure may be practiced without some specific details. In other instances, well-known methods, means, apparatus, and steps are not described in detail in order to facilitate the disclosure.

或ClustalW等本领域常用的氨基酸序列比对算法或程序容易地确定。The term "homologous protein" as used herein refers to a protein having significant similarity to the GLUT in the amino acid sequence, and the similarity in the amino acid sequence can be used by those skilled in the art, for example.

Or an amino acid sequence alignment algorithm or program commonly used in the art such as ClustalW is readily determined.

The terms "prokaryotic protein model of GLUT", "prokaryotic homologous protein of GLUT", "prokaryotic protein model", "prokaryotic protein", "prokaryotic homologous protein" and the like can be used interchangeably herein, and each represents the present disclosure. Proteins of prokaryote origin that mimic the GLUT or its specific conformer, may include wild-type proteins and mutant proteins.

The term "wild-type protein" as used herein shall be understood in the ordinary manner known to those skilled in the art and may be interpreted as a protein of an organism obtained from nature, i.e., a mutation of an amino acid residue without artificial introduction. Protein. The term "mutant protein" as used herein, refers to a protein in which a mutation in an amino acid residue is introduced into a corresponding wild-type protein. Unless otherwise specified herein, the wild type protein is represented only by the name of the protein. Unless otherwise specified, "wild type XylE protein" herein refers to a protein comprising the amino acid sequence of SEQ ID NO: 4.

The terms "mutant protein" and "a protein mutant" are used interchangeably herein to refer to a protein having a mutation in an amino acid residue on the wild type protein.

The term "transmembrane helix (TM)" as used herein denotes a portion of a helical structure spanning a cell membrane in GLUT or its prokaryotic homologous protein. In this context, the first transmembrane helix from the N-terminus of the prokaryotic homologous protein of GLUT is referred to as the first transmembrane helix, abbreviated as TM1; the second transmembrane helix from the N-terminus is referred to as the second span. Membrane spiral, abbreviated as TM2. The rest of the transmembrane helix is the same.

As used herein, "GLUT", unless otherwise indicated, indicates a group selected from the group consisting of GLUT1, GLUT2, GLUT3, GLUT4, GLUT5, GLUT6, GLUT7, GLUT8, GLUT9, GLUT10, GLUT11, GLUT12, GLUT13, GLUT14, and other GLUT subtypes. Any one or more of the proteins.

The term "large sterically hindered side chain amino acid residue mutation" as used herein refers to a mutation of an amino acid residue in an amino acid sequence of a protein substituted with an amino acid residue having a large sterically hindered side chain. The large sterically hindered side chain amino acid is selected from the group consisting of tryptophan Trp, tyrosine Tyr, phenylalanine Phe, lysine Lys, arginine Arg, glutamic acid Glu, glutamine Gln, aspartame A group consisting of acid Asp and asparagine Asn.

The term "nucleotide" as used herein, unless otherwise indicated, refers to ribonucleotides and/or deoxyribonucleotides.

As used herein, "glucose" refers to D-glucose and "xylose" refers to D-xylose.

Prokaryotic protein model of GLUT

The prokaryotic protein model of the GLUT of the present disclosure is not particularly limited as long as it is a prokaryotic homologous protein of GLUT, and is preferably a prokaryotic homologous protein derived from Escherichia coli of GLUT, and more preferably a xylose transporter XylE.

The prokaryotic protein model of the GLUT of the present disclosure is preferably a protein that mimics an outward-opening type GLUT conformer, and is preferably a mutant of a prokaryotic homologous protein of GLUT. The mutant of the prokaryotic homologous protein of the present disclosure is not particularly limited as long as it can mimic the conformational isomer of GLUT. Preferably, the mutant of the prokaryotic homologous protein is capable of mimicking the outward-opening GLUT conformer. The mutant of the prokaryotic homologous protein of the present disclosure is preferably a mutant of a prokaryotic homologous protein derived from Escherichia coli of GLUT, and more preferably a mutant of the xylose transporter XylE.

或ClustalW程序。当使用BLAST或ClustalW时，可以使用相应程序的默认参数。The prokaryotic protein model of the GLUT of the present disclosure preferably has a sequence identity of 15% or more, 16% or more, 17 or more, 18% or more, or 19% or more with the GLUT as a simulation target, and more preferably has 20% or more and 21% or more. More than 22%, 23% or more, 24% or more, 25% or more, 26% or more, 27% or more, 28% or more, 29% or more, or 30% or more. The sequence identity can be determined by one skilled in the art by using any algorithm or program known in the art and commonly used to determine the percent identity between two amino acid sequences, for example, can be used

Or ClustalW program. When using BLAST or ClustalW, you can use the default parameters of the corresponding program.

或ClustalW程序。当使用BLAST或ClustalW时，可以使用相应程序的默认参数。The prokaryotic protein model of the GLUT of the present disclosure preferably has a sequence similarity of 35% or more with the GLUT as a simulation target, and more preferably has a sequence similarity of 36% or more, 37% or more, 38% or more, or 39% or more, more preferably 40% or more, 41% or more, 42% or more, 43% or more, 44% or more or 45% or more of sequence similarity, more preferably 46% or more, 47% or more, 48% or more, 49% or more, 50% Above, 51% or more, 52% or more, or 53% or more of sequence similarity. The sequence similarity can be determined by one skilled in the art by using any algorithm or program for determining the percent similarity between two amino acid sequences known and used in the art, for example, can be used

Or ClustalW program. When using BLAST or ClustalW, you can use the default parameters of the corresponding program.

Preferably, the prokaryotic protein model of the GLUT of the present disclosure is capable of binding glucose.

Preferably, the prokaryotic protein model of the GLUT of the present disclosure has a sequence identity of more than 80% with XylE, and further preferably has a sequence identity of 81% or more, 82% or more, 83% or more, 84% or more, or 85% or more. Preferably, it has a sequence identity of 86% or more, 87% or more, 88% or more, 89% or more, or 90% or more, and more preferably has a sequence of 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more. More preferably, it has a sequence identity of 96% or more, 97% or more, 98% or more, or 99% or more, and particularly preferably 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, and 99.6. Sequence identity above %, above 99.7%, above 99.8%, above 99.9%.

Preferably, the prokaryotic protein model of the GLUT of the present disclosure is capable of transporting xylose.

The prokaryotic protein model of the present disclosure is preferably a prokaryotic protein that mimics the conformational isomer of GLUT. Preferably, the prokaryotic protein of the conformer of the mimetic GLUT comprises two or more, preferably two, three, four, five, six, seven amino acid sequences of XylE represented by SEQ ID NO: a protein of 8, 9, 10, or more amino acid sequences having a large sterically hindered side chain amino acid residue mutation. Preferably, in the mutation of the amino acid residues of the two or more large sterically hindered side chains, at least one mutation is located in the extracellular region of TM2, and at least one other mutation is located in the extracellular region selected from the extracellular region of TM1, TM5 The region of the group consisting of a portion and an extracellular region of TM8. For XylE, the extracellular region of TM2 corresponds to the amino acid residues 52 to 68 of the amino acid sequence shown in SEQ ID NO: 4, and the extracellular region of TM1 corresponds to the sequence shown by SEQ ID NO: The amino acid residues at positions 25 to 40 of the amino acid sequence, the extracellular region of TM5 corresponds to the amino acid residues from positions 172 to 190 of the amino acid sequence shown in SEQ ID NO: 4, and the extracellular region of TM8 Partially corresponds to amino acid residues from positions 311 to 326 of the amino acid sequence shown by SEQ ID NO: 4.

Specifically, the mutation of the large sterically hindered side chain amino acid residue may be, for example, a mutation produced by Gly58, Ala62, and Leu65 located in the extracellular region of TM2; Ala29 and Ser32 located in the extracellular region of TM1. , Glu36; Leu176 located in the extracellular region of TM5; Leu315, Thr318, Ile319, Gly322 located in the extracellular region of TM8, wherein the amino acid number corresponds to the amino acid sequence of the amino acid sequence of XylE shown in SEQ ID NO: . Preferably, the large sterically hindered side chain amino acid is selected from the group consisting of tryptophan, tyrosine, phenylalanine, lysine, arginine, glutamic acid, glutamine, aspartic acid, aspartic acid The group of the amide composition is more preferably selected from the group consisting of tryptophan, tyrosine and phenylalanine, and more preferably tryptophan.

Specifically, in the above two or more large sterically hindered side chain amino acid residue mutations, preferably, at least one of the mutations is located at a site selected from the group consisting of the 58th, 62nd, and 65th positions, at least one other mutation Located at a site selected from the group consisting of the 29th, 32nd, 36th, 176th, 315th, 318th, 319th, and 322th positions. Preferably, at least one of the mutations is selected from the group consisting of Gly58Trp (the label indicates that the glycine at position 58 is replaced by tryptophan, the same applies hereinafter), Ala62Trp and Leu65Trp, and at least one other mutation is selected from the group consisting of Ala29Trp, Ser32Trp, Glu36Trp, Leu176Trp, A group consisting of Leu315Trp, Thr318Trp, Ile319Trp, Gly322Trp. The amino acid number therein corresponds to the number of the amino acid in the amino acid sequence of XylE shown in SEQ ID NO: 4.

It should be noted that, in addition to the amino acid mutations at the specific sites mentioned above, the prokaryotic protein model of the GLUT of the present disclosure may further comprise a mutation that adds, deletes and/or replaces one or more amino acids at other sites, as long as the Mutation does not affect the nature of the prokaryotic protein mimicking GLUT, for example, without affecting the binding of the prokaryotic protein to glucose, is included within the scope of the present disclosure. For prokaryotic proteins that mimic the conformational isomer of GLUT, as long as the above-described mutations at other sites do not affect the conformation of the mimetic outward-opening GLUT protein conformer possessed by the prokaryotic protein, it is included in the present disclosure. Within the scope. As an example thereof, physical properties and chemical properties which are generated outside the conserved sites of prokaryotic proteins or a similar substitution between two amino acids may be mentioned. As another example, one, two, three, four, five, six, seven, eight, nine or ten or more generated outside the conserved site of the prokaryotic protein may be mentioned. Addition and/or deletion of amino acids, any one of the two, two, three, four, five, six, seven, eight, nine or more amino acids may be Neighbor or not adjacent. Figure 2 shows the conserved site of XylE, the position of which is shown in Figure 2 for XylE and The alignment of the amino acid sequences of GLUT1, GLUT2, GLUT3, and GLUT4 is indicated by "*" below.

The prokaryotic protein model of the GLUT of the present disclosure is, for example, the following proteins (a) to (x):

(a) comprising the amino acid sequence shown in SEQ ID NO: 4, and in the amino acid sequence shown in SEQ ID NO: 4, the glycine at position 58 is replaced with tryptophan, and the leucine at position 315 is replaced with tryptamine An acid protein; or a protein comprising the amino acid sequence of SEQ ID NO: 6.

(b) comprising the amino acid sequence shown in SEQ ID NO: 4, and in the amino acid sequence shown in SEQ ID NO: 4, the glycine at position 58 is replaced with tryptophan, and the alanine at position 29 is replaced with tryptamine An acid protein; or a protein comprising the amino acid sequence of SEQ ID NO: 8.

(c) comprising the amino acid sequence of SEQ ID NO: 4, and in the amino acid sequence shown in SEQ ID NO: 4, the glycine at position 58 is replaced with tryptophan, and the serine at position 32 is replaced with tryptophan. a protein; or a protein comprising the amino acid sequence of SEQ ID NO: 10.

(d) comprising the amino acid sequence shown in SEQ ID NO: 4, and in the amino acid sequence shown in SEQ ID NO: 4, the glycine at position 58 is replaced with tryptophan, and the glutamic acid at position 36 is replaced with tryptamine An acid protein; or a protein comprising the amino acid sequence of SEQ ID NO: 12.

(e) comprising the amino acid sequence shown in SEQ ID NO: 4, and in the amino acid sequence shown in SEQ ID NO: 4, the glycine at position 58 is replaced with tryptophan, and the threonine at position 318 is replaced with tryptamine. An acid protein; or a protein comprising the amino acid sequence of SEQ ID NO: 14.

(f) comprising the amino acid sequence shown in SEQ ID NO: 4, and in the amino acid sequence shown in SEQ ID NO: 4, the glycine at position 58 is replaced with tryptophan, and the isoleucine at position 319 is substituted with color. A protein of a tyrosine; or a protein comprising the amino acid sequence of SEQ ID NO: 16.

(g) comprising the amino acid sequence shown in SEQ ID NO: 4, and in the amino acid sequence shown in SEQ ID NO: 4, the glycine at position 58 is replaced with tryptophan, and the glycine at position 322 is replaced with tryptophan. a protein; or a protein comprising the amino acid sequence of SEQ ID NO: 18.

(h) comprising the amino acid sequence shown in SEQ ID NO: 4, and in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan and the alanine at position 29 is replaced with A protein of tryptophan; or a protein comprising the amino acid sequence of SEQ ID NO: 20.

(i) comprising the amino acid sequence shown in SEQ ID NO: 4, and in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan, and the serine at position 32 is replaced with tryptamine. An acid protein; or a protein comprising the amino acid sequence of SEQ ID NO: 22.

(j) comprising the amino acid sequence shown in SEQ ID NO: 4, and in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan and the glutamic acid at position 36 is replaced with A protein of tryptophan; or a protein comprising the amino acid sequence of SEQ ID NO: 24.

(k) comprising the amino acid sequence shown in SEQ ID NO: 4, and in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan and the leucine at position 176 is replaced with A protein of tryptophan; or a protein comprising the amino acid sequence of SEQ ID NO: 26.

(l) comprising the amino acid sequence shown in SEQ ID NO: 4, and in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is substituted with tryptophan and the leucine at position 315 is replaced with A protein of tryptophan; or a protein comprising the amino acid sequence of SEQ ID NO: 28.

(m) comprising the amino acid sequence shown in SEQ ID NO: 4, and in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan and the threonine at position 318 is replaced with A protein of tryptophan; or a protein comprising the amino acid sequence of SEQ ID NO:30.

(n) comprising the amino acid sequence shown in SEQ ID NO: 4, and in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan and the isoleucine at position 319 is replaced. A protein that is tryptophan; or a protein that includes the amino acid sequence of SEQ ID NO:32.

(o) comprising the amino acid sequence shown in SEQ ID NO: 4, and in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan, and the glycine at position 322 is replaced with tryptophan. An acid protein; or a protein comprising the amino acid sequence of SEQ ID NO:34.

(p) comprising the amino acid sequence shown in SEQ ID NO: 4, and in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan and the alanine at position 29 is replaced with A protein of tryptophan; or a protein comprising the amino acid sequence of SEQ ID NO: 36.

(q) comprising the amino acid sequence shown in SEQ ID NO: 4, and in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan, and the serine at position 32 is replaced with tryptamine An acid protein; or a protein comprising the amino acid sequence of SEQ ID NO: 38.

(r) comprising the amino acid sequence shown in SEQ ID NO: 4, and in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with a color A protein in which a tyrosine at position 36 is substituted with tryptophan or a protein having an amino acid sequence as shown in SEQ ID NO: 40.

(s) comprising the amino acid sequence shown in SEQ ID NO: 4, and in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan and the leucine at position 176 is replaced with A protein of tryptophan; or a protein comprising the amino acid sequence of SEQ ID NO: 42.

(t) comprising the amino acid sequence shown in SEQ ID NO: 4, and in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan and the leucine at position 315 is replaced with A protein of tryptophan; or a protein comprising the amino acid sequence of SEQ ID NO: 44.

(u) includes the amino acid sequence shown in SEQ ID NO: 4, and in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan and the threonine at position 318 is replaced with A protein of tryptophan; or a protein comprising the amino acid sequence of SEQ ID NO:46.

(v) comprising the amino acid sequence shown in SEQ ID NO: 4, and in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan and the isoleucine at position 319 is replaced. A protein that is tryptophan; or a protein comprising the amino acid sequence of SEQ ID NO:48.

(w) comprising the amino acid sequence shown in SEQ ID NO: 4, and in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan, and the glycine at position 322 is replaced with tryptamine An acid protein; or a protein comprising the amino acid sequence of SEQ ID NO: 50.

(x) comprising the amino acid sequence shown in SEQ ID NO: 4, and in the amino acid sequence shown in SEQ ID NO: 4, the glycine at position 58 is replaced with tryptophan, and the leucine at position 176 is replaced with tryptamine An acid protein; or a protein comprising the amino acid sequence of SEQ ID NO: 68.

The prokaryotic protein model of the GLUT of the present disclosure may also be affixed to a label useful for isolation, purification or identification, detection at its N-terminal side and/or C-terminal side, such as an oligopeptide represented by polyhistidine. . There is no particular limitation on the length and structure of the label as long as it does not impair the properties of the prokaryotic protein as a model of the GLUT, such as substrate binding and conformation. When the aforementioned tag is added to the prokaryotic protein of the present disclosure, for example, a polynucleotide encoding the aforementioned tag can be prepared and genetically added to the end of the polynucleotide encoding the prokaryotic protein of the present disclosure using a method known to those skilled in the art. The aforementioned label can also be chemically combined and appended to the prokaryotic proteins of the present disclosure.

Method for preparing prokaryotic protein model of GLUT

As an example of a method for producing a nucleotide sequence encoding a prokaryotic protein of the present disclosure, it is exemplified:

(I) a method of artificially synthesizing a polynucleotide comprising the nucleotide sequence by converting an amino acid sequence of a prokaryotic protein of the present disclosure into a nucleotide sequence; or

(II) preparing a polynucleotide comprising a whole or a partial sequence encoding the prokaryotic protein from a cDNA of a prokaryotic protein or the like using a DNA amplification method such as polymerase chain reaction (PCR), and preparing the multinuclear compound by an appropriate method. A method in which a glycoside is linked.

In the case of introducing a mutation into the polynucleotide of the present disclosure, a method known to those skilled in the art can be used. As an example, a method of performing PCR using a primer having a mutation can be used. The site into which the mutation is introduced may, for example, be a site selected from the group consisting of TM2, TM1, TM5 and/or TM8 of the prokaryotic homologous protein of the GLUT, preferably located in the extracellular region selected from the group consisting of TM2, TM1, TM5 and/or TM8 The location.

The host which expresses the prokaryotic protein of the present disclosure is not particularly limited as long as it can express the protein of the present disclosure in a certain yield, and for example, Escherichia coli (JM109 strain, BL21 (commercially available) can be mentioned. DE3) strain, W3110 strain, etc.), Bacillus subtilis. It is further preferred to use E. coli as a host.

In the case of using a polynucleotide encoding a prokaryotic protein of the present disclosure to transform a host, the polynucleotide itself can be used, and it is more preferable to use an appropriate one in an expression vector (for example, a plasmid which is usually used for transformation of prokaryotic cells, etc.). An expression vector for the polynucleotide of the present disclosure is inserted at a site. The expression vector is not particularly limited as long as it can stably exist and replicate in the transformed host. In the case of using Escherichia coli as a host, a commercially available pET plasmid vector, pUC plasmid vector can be exemplified. , pTrc plasmid vector, pCDF plasmid vector, pBBR plasmid vector.

In order to transform a host using an expression vector inserted with the polynucleotide of the present disclosure prepared by the aforementioned method, a method known to those skilled in the art (for example, the method described in Molecular Cloning, Cold Spring Harbor Laboratory, 256, 1992) can be used. get on. The transformant obtained by the above-described method can be screened by an appropriate method, for example, by screening with a drug resistance gene carried on an expression vector, thereby obtaining an expression capable. A transformant of a prokaryotic protein disclosed.

In order to prepare the expression vector of the present disclosure from the transformant of the present disclosure, it can be prepared by extracting the expression vector of the present disclosure from the transformant of the present disclosure by a method suitable for the host used in the transformation. The extraction can be carried out using an alkaline extraction method or any commercial kit commonly used in the art.

By culturing the transformant of the present disclosure, the prokaryotic protein of the present disclosure is recovered from the obtained culture (including a host cell and a culture medium in which the expression vector of the present disclosure is transformed), whereby the prokaryotic protein of the present disclosure can be produced. The above culture may be carried out using a medium and culture conditions suitable for the host used in the transformation. In order to selectively proliferate a transformant into which the expression vector of the present disclosure has been introduced, it is preferred to cultivate a drug corresponding to the drug resistance gene contained in the expression vector to the culture medium. The medium may also contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cysteamine, thioglycolate, and dithiothreitol as needed. Specifically, in the case where the host is Escherichia coli, the culture medium may be a LB (Luria-Bertani) medium supplemented with a necessary nutrient source for the wild type or mutant XylE protein, and the culture temperature may be 10 ° C to 40 ° C. °C, preferably 20 ° C to 37 ° C, more preferably about 25 ° C, the pH of the medium is from pH 6.0 to pH 8.5, preferably around pH 7.0. Further, in the case where the vector of the present disclosure contains an inducible promoter, it is preferred to carry out the induction under the condition that the prokaryotic protein of the present disclosure can be expressed well. As the inducer, IPTG (isopropyl-β-D-thiogalactopyranoside, isopropyl-β-D-thiogalactoside) can be exemplified. When the host is Escherichia coli, the turbidity (absorbance at 600 nm) of the culture solution is measured, and when it is about 1.0 to 2.0, an appropriate amount of IPTG is added, and then the culture is continued. The concentration of IPTG added may be appropriately selected from the range of 0.01 to 1.0 mmol/L, preferably in the range of 0.1 to 0.5 mmol/L. The various conditions associated with IPTG induction may be carried out according to conditions well known in the art.

In order to recover the prokaryotic protein of the present disclosure from the culture obtained by culturing the transformant of the present disclosure, separation/purification can be carried out from the culture using a method suitable for the expression pattern of the prokaryotic protein of the present disclosure in the transformant. For example, after the cells are separated and collected by centrifugation, the cells are disrupted by adding an enzyme treatment agent, a surfactant, or the like, or using ultrasonic waves, a French press, or the like, and the protein of the present disclosure is extracted and then purified. For the broken cells, preliminary separation of the components can be carried out by centrifugation.

In order to purify the prokaryotic protein of the present disclosure, a method known in the art may be used. As an example, purification using a centrifugation method is mentioned. In the centrifugation method, there are ultracentrifugation method, differential centrifugation method, density gradient centrifugation method, etc., and the purification operation can also be carried out by combining these centrifugation methods. As another example, purification using liquid chromatography can be mentioned. In the liquid chromatography, there are ion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, affinity chromatography, and the like, and purification operations can also be carried out by combining these chromatography methods. Those skilled in the art can also carry out purification using a combination of centrifugation and liquid chromatography as needed. Since the prokaryotic protein of the present disclosure is a membrane protein, it is preferred to include in the extraction a step of extracting the protein of the present disclosure from the cell membrane component using a detergent, for example, a dodecyl-β-D- Maltoside (DDM).

In order to confirm the presence, quality or purity of the resulting prokaryotic protein of the present disclosure, it can be carried out using techniques well known to those skilled in the art. For example, molecular exclusion chromatography, electrophoresis, enzyme-linked immunosorbent assay (ELISA), immunoblotting (such as Western blotting), and the like can be mentioned.

Furthermore, structural information of the proteins of the present disclosure can be obtained by X-ray crystallographic studies. Specifically, the protein of the present disclosure is crystallized by an evaporation-diffusion equilibrium crystallization method of a hanging drop at a suitable protein concentration. The crystals of the prokaryotic proteins of the present disclosure can be obtained by optimization of crystallization conditions (including precipitants and their concentrations, pH buffers, salts, additives, detergents, crystallization time, etc.) by methods well known to those skilled in the art. After the diffraction data is collected by an X-ray crystal diffraction apparatus (for example, SSRF BL17U harness station), integral calculation of diffraction data, molecular replacement of integration results, construction of a structural model, and structural correction are performed by software commonly used by those skilled in the art, thereby Analyze the structure of the protein.

Method for screening binders for GLUT

The screening method of the present disclosure is characterized in that a prokaryotic protein model simulating GLUT is used to screen for a binding agent that can bind to GLUT. Preferably, the screening methods of the present disclosure employ prokaryotic proteins that mimic specific GLUT conformers to screen for binding agents that can bind to the particular GLUT conformer. Further preferably, the screening methods of the present disclosure employ a prokaryotic protein that mimics the outwardly opening GLUT conformer to screen for binding to an outwardly opening GLUT conformer.

Preferably, the screening method of the present disclosure employs a first prokaryotic protein that mimics the GLUT and a second prokaryotic protein that mimics a specific GLUT conformer, and by analyzing differences in the binding of the candidate molecule to the two, the screening can be combined with A binding agent for a different conformational isomer of the GLUT conformer. Further preferably, the screening method of the present disclosure employs a first prokaryotic protein that mimics the GLUT and a second prokaryotic protein that mimics the outwardly opening GLUT conformer, and the screening can be combined by analyzing the difference in binding of the candidate molecule to the two. A binding agent for the inwardly opening GLUT conformer.

The method of the present disclosure preferably employs XylE as the first prokaryotic protein. Further, it is preferred to use a mutant XylE having a large sterically hindered side chain amino acid residue mutation as the second prokaryotic protein. It is further preferred to employ a large sterically hindered side chain amino acid residue mutation in a region of the extracellular region of TM2 and a region selected from the group consisting of the extracellular region of TM1, the extracellular region of TM5, and the extracellular region of TM8. The mutant XylE acts as a second prokaryotic protein. The number of amino acid residue mutations in the large sterically hindered side chain is preferably two or more, and more preferably two, three, four, five, six, seven, eight, nine, ten or 11 or more. The large sterically hindered side chain amino acid is preferably selected from the group consisting of tryptophan, tyrosine, phenylalanine, lysine, arginine, glutamic acid, glutamine, aspartic acid, and asparagine. The amino acid of the composition group is more preferably an amino acid selected from the group consisting of tryptophan, tyrosine, and phenylalanine, and more preferably tryptophan.

It should be noted that, as a prokaryotic protein simulating GLUT, in addition to XylE, if other prokaryotic-derived homologous proteins using GLUT can achieve the object of the present disclosure according to the concept and technical flow of the present disclosure, it also belongs to the present disclosure. range.

The method of the present disclosure for screening a binding agent for GLUT preferably comprises the steps of 1) and/or 2) below:

1) a step of detecting the binding of the first prokaryotic protein to the candidate molecule;

2) a step of detecting the binding of the second prokaryotic protein to the candidate molecule;

The screening method of the present disclosure may further include the following steps of 3)

3) A step of determining the binding of the candidate molecule on the GLUT based on the detection results of step 1) and/or step 2). According to the binding condition judged in the step 3), it can be judged whether or not the candidate molecule is a binding agent for the GLUT.

Among them, the first prokaryotic protein of step 1) is preferably a prokaryotic protein model of GLUT. The second prokaryotic protein of step 2) is preferably a protein comprising an amino acid sequence having a large sterically hindered side chain amino acid residue mutation in the amino acid sequence of the aforementioned first prokaryotic protein. The second prokaryotic protein of step 2) is also preferably a prokaryotic protein that mimics the exo-opening type GLUT conformer.

Wherein, the determination of the binding of the candidate molecule on the GLUT in step 3) is preferably such that if the binding of the candidate molecule to the first prokaryotic protein is detected in step 1), it is determined that the candidate molecule can bind to the GLUT, ie, the candidate The molecule is a binding agent to the GLUT. The determination of the binding of the candidate molecule on the GLUT in step 3) is also preferably to determine which conformer of the GLUT can be bound by the candidate molecule by comparing the binding result of the first prokaryotic protein and the second prokaryotic protein with the candidate molecule. .

For the judgment in the above step 3), preferably, in the case where the protein which mimics the outward-opening type GLUT conformer is used as the second prokaryotic protein in the step 2), if the candidate molecule is detected in the step 2) Binding of the second prokaryotic protein, it is judged that the candidate molecule can bind to the outward-opening GLUT conformer; if only the binding of the candidate molecule to the first prokaryotic protein is detected in step 1), no detection is detected in step 2) The binding of the candidate molecule to the second prokaryotic protein determines that the candidate molecule is capable of binding to the inwardly opening GLUT conformer. The second prokaryotic protein mimicking the exo-opening type GLUT conformer is preferably a prokaryotic protein having a large sterically hindered side chain amino acid residue mutation.

The candidate molecule for the binding agent of GLUT may be any natural or artificial molecule, and may be any small molecule or macromolecule, such as a chemical molecule or a biomolecule, including a chemical small molecule, a chemical macromolecule, a biological macromolecule or the like. A candidate molecule for a binding agent to GLUT can be an antibody molecule, and as an antibody molecule, can be an intact antibody, or an antibody fragment. A fragment of an intact antibody refers to a region of a part of the aforementioned intact antibody, for example, a monoclonal antibody or a polyclonal antibody, and examples thereof include Fab, Fab', F(ab') 2, Fv (antibody variable region), and single Chain antibodies (H chain, L chain, H chain V region and L chain V region, etc.), scFv, diabody (scFv dimer), dsFv (disulfide stabilized V region), at least partially complementary Peptides, Nanobodies, etc. of the complementarity determining region (CDR).

A candidate molecule for a binding agent to GLUT may be a molecule known to have some pharmaceutically or biological activity, or a molecule that has not been demonstrated to have any pharmaceutical or biological activity. Any commercially available small molecule or macromolecular library can be used as a library of candidate molecules for binding agents to GLUT. Those skilled in the art can also construct a library comprising any small molecule or macromolecule as needed, and screen the binding agent for GLUT using the screening method of the present disclosure.

The sequence of steps 1) and 2) is not particularly limited as long as the binding result of the first prokaryotic protein to the candidate molecule and the binding result of the second prokaryotic protein to the candidate molecule can be separately obtained, and steps 1) and 2) are also This can be done, for example, synchronously or simultaneously.

Wherein, the above step 1) may include the following sub-steps:

1-1) a step of detecting whether the first prokaryotic protein binds to the candidate molecule;

1-2) a step of detecting the binding force of the first prokaryotic protein to the candidate molecule.

The above step 2) may include the following sub-steps:

2-1) a step of detecting whether the second prokaryotic protein binds to the candidate molecule;

2-2) A step of detecting the binding force of the second prokaryotic protein to the candidate molecule.

The order of steps 1-1) and 1-2) is not particularly limited as long as the result of the binding of the first prokaryotic protein to the candidate molecule and the binding force of the two are obtained, and steps 1-1) and steps are not particularly limited. 1-2) can also be carried out, for example, simultaneously or simultaneously. The order of steps 2-1) and 2-2) is not particularly limited as long as the result of the binding of the second prokaryotic protein to the candidate molecule and the binding force of the two are obtained, and step 2-1) and the step are not particularly limited. 2-2) can also be carried out, for example, simultaneously or simultaneously.

As a method of detecting the binding of the prokaryotic protein of the present disclosure to the candidate molecule, there is no particular limitation as long as it is a method capable of measuring the magnitude of binding and/or binding force. Preferably, the detection is carried out in a solution environment. Specific examples of the method include an isothermal calorimetric method, a microcalorimetric method, a surface plasmon resonance method, and the like, and an indirect detection of binding by detecting the influence of the candidate molecule on the transport activity of the prokaryotic protein of the present disclosure. Methods such as liposome transport assays. Preferably, the detection of binding can be carried out by using a liposome transport assay, and the magnitude of the binding force can be determined by isothermal calorimetry or microcalorimetry. The detection methods used in steps 1) and 2) may be the same or different. The detection methods employed in any two of steps 1-1), 1-2), 2-1) and 2-2) may be the same or different.

In a preferred embodiment, the method of the present disclosure determines whether a candidate molecule binds to and/or binds to a first prokaryotic protein or a second prokaryotic protein using isothermal calorimetry and/or microcalorimetry. In another preferred embodiment, the first prokaryotic protein is a protein comprising the amino acid sequence of SEQ ID NO: 4, and the second prokaryotic protein is one or more of the XylEs described in the aforementioned " Prokaryotic Protein Model of GLUT " section. mutant.

In another preferred embodiment, the methods of the present disclosure further comprise the step of detecting the effect of the candidate molecule on the transport activity of the prokaryotic protein mimicking the GLUT by a liposome transport assay. Based on the results of the liposome transport assay, the effect of the candidate molecule on the transport activity of the GLUT can be judged. For example, if the candidate molecule inhibits the transport activity of the prokaryotic protein model of the GLUT, it is judged that the candidate molecule can inhibit the transport activity of the GLUT. The prokaryotic protein of the mimetic GLUT utilized in the liposome transport assay may be the same as or different from the first prokaryotic protein in step 1). The sequence between the steps of the liposome transport experiment and any of the steps 1), 2) and 3) is not particularly limited, and may also be, for example, in steps 1), 2) and 3) Any of the steps are performed synchronously or simultaneously.

For Isothermal Titration Calorimetry (ITC), reference can be made to Non-Patent Document 9 in a manner well known to those skilled in the art. In an exemplary embodiment, the protein having a concentration between 10 and 1000 μmol/L is added to the reaction cell of the isothermal calorimeter at a reaction temperature of 15 to 30 ° C, and the concentration is 0.1 mmol/ The substrate or candidate molecule of L ~ 100 mmol / L was titrated to determine the binding force data between the two.

For Microscale Thermophoresis (MST), refer to Parker et al. (Joanne L. Parker & Simon Newstead, Molecular basis of nitrate uptake by the plant nitrate transporter NRT l.1, Nature 507, 68-72, March 2014, The method described by doi: 10.1038/nature 13116) is carried out in a manner well known to those skilled in the art. In an exemplary embodiment, protein is used at a concentration between 500 and 50,000 nmol/L, and a 1:1 gradient is applied to a substrate or candidate molecule having a starting concentration between 10 and 10000 μmol/L. The substrate was mixed with a gradient diluted substrate or candidate molecule, and then the binding force was measured using a micro thermophoresis.

For the liposome transport assay, reference can be made to Non-Patent Document 9 in a manner well known to those skilled in the art. In a preferred embodiment, liposomes carrying the prokaryotic proteins of the present disclosure are first prepared using a nitrogen-dried polar lipid of E. coli, followed by addition to the proteoliposome at 25 °C. Specific concentration of candidate binder molecules, after incubation, add 1 μCi of ³ H-labeled substrate to 100 μl of KPM (50 mmol/L potassium phosphate buffer pH 6.5, 2 mmol/L magnesium chloride), then add 2 μl to the solution. The proteoliposome, after 30 s of reaction, the solution containing the proteoliposome was filtered through a 0.22 μm filter, then the filter was rinsed with 2 ml of KPM solution, and finally the filter was added to 500 μl of scintillation fluid and incubated overnight. After reading with the counter.

The screening methods of the present disclosure can be used to screen for binding agents to GLUT, such as binding agents that inhibit the transport activity of GLUT. The binding agent that inhibits the transport activity of GLUT may also be referred to as an inhibitor of the transport activity of GLUT, or a GLUT inhibitor. Preferably, the screening methods of the present disclosure can be used to screen for drugs against GLUT, such as drugs for treating cancer, which are cancers associated with overexpression of GLUT. The cancer includes, but is not limited to, lymphoma, colorectal cancer, hepatocellular carcinoma, head and neck cancer, stomach cancer, prostate cancer, thyroid cancer, kidney cancer, lung cancer, pancreatic cancer, sarcoma, laryngeal cancer, esophageal cancer, brain cancer, breast cancer. , choriocarcinoma, ovarian cancer, endometrial cancer, retinoblastoma, rhabdomyosarcoma, glioma, cervical cancer, gallbladder cancer, oral cancer, squamous cell carcinoma, bladder cancer, multiple myeloma, melanoma, Testicular seminoma and the like. Screening of the GLUT inhibitor or drug can be carried out by methods and procedures similar to those described above for screening for binding agents to GLUT.

Kit for screening binding agents for GLUT

The kit for screening a binding agent for GLUT of the present disclosure is not particularly limited as long as it contains a prokaryotic protein that mimics GLUT. The kit of the present disclosure preferably comprises a prokaryotic protein that mimics a specific conformer of the GLUT. Preferably, the kit of the present disclosure comprises a prokaryotic protein that mimics an outwardly open GLUT conformer.

The kit of the present disclosure preferably comprises a first prokaryotic protein that mimics the GLUT and a second prokaryotic protein that mimics a particular conformer of the GLUT. Preferably, the second prokaryotic protein mimics an outward open-type GLUT conformer. The first prokaryotic protein and the second prokaryotic protein comprised by the kit of the present disclosure may be a prokaryotic protein simulating a GLUT and a prokaryotic protein mimicking a specific conformer of the GLUT, respectively, as described in any part of the disclosure, wherein the second prokaryotic protein Prokaryotic proteins that mimic the outwardly open conformational conformation of the GLUT can be described in any part of the disclosure.

The first prokaryotic protein contained in the kit of the present disclosure is preferably a protein comprising the amino acid sequence of SEQ ID NO: 4. The second prokaryotic protein contained in the kit is preferably a protein comprising a sequence having a large sterically hindered side chain amino acid residue mutation in the amino acid sequence of the first prokaryotic protein. Preferably, a mutant protein having a large sterically hindered side chain amino acid residue mutation mimics an outwardly opening GLUT conformer.

Further preferably, the kit of the present disclosure comprises a large space in the extracellular region portion of TM2 and a region selected from the group consisting of the extracellular region portion of TM1, the extracellular region portion of TM5, and the extracellular region portion of TM8 A mutant of the first prokaryotic protein mutated by the amino acid residue of the side chain is used as the second prokaryotic protein. The number of amino acid residue mutations in the large sterically hindered side chain is preferably two or more, and more preferably two, three, four, five, six, seven, eight, nine, ten or eleven More than one. The large sterically hindered side chain amino acid is preferably selected from the group consisting of tryptophan, tyrosine, phenylalanine, lysine, arginine, glutamic acid, glutamine, aspartic acid, and asparagine. The amino acid of the composition group is more preferably an amino acid selected from the group consisting of tryptophan, tyrosine, and phenylalanine, and more preferably tryptophan. The second prokaryotic protein comprised by the kit of the present disclosure is preferably one or more of the XylE mutants described in the " Prokaryotic Protein Model of GLUT " section above.

It should be noted that, as a prokaryotic protein of the mimetic GLUT included in the kit of the present disclosure, in addition to using XylE, if other prokaryotic-derived homologous proteins of GLUT are used, the present disclosure can also realize the present disclosure. The purpose is also within the scope of the present disclosure.

The kits of the present disclosure can be used to screen for binding agents to GLUT, such as binding agents that inhibit the transport activity of GLUT. The inhibition of GLUT rotation The active binding agent may also be referred to as an inhibitor of the transport activity of GLUT, or a GLUT inhibitor. Preferably, the kits of the present disclosure can be used to screen for drugs against GLUT, such as drugs for treating cancer, which are cancers associated with overexpression of GLUT. The cancer includes, but is not limited to, lymphoma, colorectal cancer, hepatocellular carcinoma, head and neck cancer, stomach cancer, prostate cancer, thyroid cancer, kidney cancer, lung cancer, pancreatic cancer, sarcoma, laryngeal cancer, esophageal cancer, brain cancer, breast cancer. , choriocarcinoma, ovarian cancer, endometrial cancer, retinoblastoma, rhabdomyosarcoma, glioma, cervical cancer, gallbladder cancer, oral cancer, squamous cell carcinoma, bladder cancer, multiple myeloma, melanoma, Testicular seminoma and the like.

The kit of the present disclosure may comprise, in addition to the prokaryotic proteins of the present disclosure, reagents, tools and/or devices for detecting binding of prokaryotic proteins of the present disclosure to candidate molecules. Preferably, the kit of the present disclosure comprises reagents, tools and/or devices for detecting binding of a prokaryotic protein of the present disclosure to a candidate molecule by microcalorimetry. Optionally, one or more control samples may be included in the kit, and the control sample may be a positive control or a negative control sample.

Regardless of the form of the kit, one or more containers containing the reagents therein and preferably suitably aliquoted are typically included. The components of the kit may be packaged in an aqueous medium or in lyophilized form. The kit may also contain one or more excipients, diluents and/or carriers. Non-limiting examples of excipients, diluents, and/or carriers include water, buffers, physiological saline.

The kit may also include instructions for using the kit components as well as any other reagents not included in the kit. Additionally, the kit is not limited to the particular items identified above and may comprise any reagent for manipulating or characterizing the binding of the prokaryotic proteins of the present disclosure to the candidate molecule.

Method for immobilizing conformational isomers of prokaryotic homologous proteins of GLUT

The method of the present disclosure for immobilizing a conformer of a prokaryotic homologous protein of GLUT is characterized in that the conformation of the prokaryotic homologous protein is immobilized by introducing a mutation of an amino acid residue into a prokaryotic homologous protein of GLUT. Preferably, the conformation of the prokaryotic homologous protein is immobilized by introducing a large sterically hindered side chain amino acid residue mutation into the prokaryotic homologous protein of GLUT. Further preferably, the conformation of the prokaryotic homologous protein is fixed to an outward open conformation by introducing a large sterically hindered side chain amino acid residue mutation into the prokaryotic homologous protein of GLUT.

Specifically, the method of immobilizing the prokaryotic homologous protein conformation of GLUT of the present disclosure preferably introduces two or more, preferably two, three, four, five, six, seven, eight on the homologous prokaryotic protein. One, nine, ten or more large sterically hindered side chain amino acid residue mutations. Preferably, at least one mutation in the amino acid residue mutation of the two or more large sterically hindered side chains is located in the extracellular region of the prokaryotic protein of TM2, and at least one other mutation is located in the extranuclear protein selected from the extracellular domain of TM1. A region of the group consisting of the region portion, the extracellular region portion of TM5, and the extracellular region portion of TM8.

Preferably, the method of the present disclosure for immobilizing the conformational isomer of a prokaryotic homologous protein of GLUT comprises the steps of:

1) introducing a large sterically hindered side chain amino acid residue mutation in the extracellular region of TM2 of the prokaryotic homologous protein;

2) A large sterically hindered side chain amino acid residue mutation is introduced in a region of the prokaryotic homologous protein selected from the group consisting of the extracellular region portion of TM1, the extracellular region portion of TM5, and the extracellular region portion of TM8.

The order of steps 1) and 2) is not particularly limited as long as the above-mentioned mutation can be introduced, and steps 1) and 2) can also be carried out, for example, simultaneously or simultaneously.

Specifically, the mutation of the large sterically hindered side chain amino acid residue may be, for example, a mutation produced by Gly58, Ala62, and Leu65 located in the extracellular region of TM2; Ala29 and Ser32 located in the extracellular region of TM1. , Glu36; Leu176 located in the extracellular region of TM5; Leu315, Thr318, Ile319, Gly322 located in the extracellular region of TM8. The amino acid number therein corresponds to the number of the amino acid in the sequence of XylE shown in SEQ ID NO: 4. For prokaryotic homologous proteins of other GLUTs other than XylE, one of skill in the art can readily confirm the corresponding position of the above site on the prokaryotic homologous protein using any known and commonly used amino acid sequence alignment algorithm or program.

The large sterically hindered side chain amino acid is preferably selected from the group consisting of tryptophan, tyrosine, phenylalanine, lysine, arginine, glutamic acid, glutamine, aspartic acid, and asparagine. The group is further preferably selected from the group consisting of tryptophan, tyrosine and phenylalanine, and most preferably tryptophan.

Specifically, at least one mutation is located at the 58th, 62nd, and 65th The site of the group consisting of at least one other mutation is located in a group selected from the group consisting of the 29th, 32nd, 36th, 176th, 315th, 318th, 319th, and 322th positions. Site. Preferably, wherein at least one mutation is selected from the group consisting of Gly58Trp, Ala62Trp and Leu65Trp, and at least one other mutation is selected from the group consisting of Ala29Trp, Ser32Trp, Glu36Trp, Leu176Trp, Leu315Trp, Thr318Trp, Ile319Trp, Gly322Trp. The amino acid number therein corresponds to the number of the amino acid in the sequence of XylE shown in SEQ ID NO: 4. Those skilled in the art can readily determine the corresponding positions of these sites on prokaryotic homologous proteins of other GLUTs other than XylE as above.

It should be noted that in addition to the amino acid mutations at the specific sites described above, the methods of the present disclosure may further comprise introducing mutations at other sites as long as the mutation does not affect the fixation of the conformation of the prokaryotic homologous protein of GLUT. As an example thereof, physical properties and chemical properties which are generated outside the conserved sites of prokaryotic proteins or a similar substitution between two amino acids may be mentioned. As another example, one, two, three, four, five, six, seven, eight, nine or ten or more generated outside the conserved site of the prokaryotic protein may be mentioned. Addition and/or deletion of amino acids, any one of the two, two, three, four, five, six, seven, eight, nine or more amino acids may be Neighbor or not adjacent.

To introduce the above mutation method, a method known in the art can be used. For example, in order to obtain a polynucleotide encoding a mutant protein, a primer having a nucleotide mutation can be used, and a polynucleotide encoding a desired prokaryotic protein (such as cDNA) can be used as a template for PCR; or a target mutant prokaryotic protein can be used. The amino acid sequence is converted into a nucleotide sequence, and a polynucleotide comprising the nucleotide sequence is artificially synthesized. Thereafter, by ligating the polynucleotide into an appropriate expression vector, and transforming the expression vector into an appropriate host, the host transformed with the expression vector is cultured under appropriate conditions to express and obtain a conformation-fixed mutation. Type protein. The specific operation can be carried out by those skilled in the art using conventional methods in the art, as described above.

Hereinafter, the embodiments are illustrated in further detail to explain the present disclosure, and the present disclosure is not limited to the embodiments.

Example 1

Preparation of XylE protein

Use primers with the following sequence:

5' start-end forward primer GATGCACATATGAATACCCAGTATAATTCCAG,

The 3'-end reverse primer CGGATCCTCGAGTTACAGCGTAGCAGTTTGTTGTG amplifies the full-length DNA encoding the XylE protein, and the DNA sequence was confirmed by sequencing to be the sequence shown in SEQ ID NO: 3.

The DNA encoding the XylE protein was cloned into the pET15b vector (Novagen) by molecular cloning means, and the vector was transformed into Escherichia coli BL21 (DE3) strain, and XylE protein was expressed by using an expression system of Escherichia coli BL21 (DE3) strain, which has The amino acid sequence is shown in SEQ ID NO: 4.

Specifically, an initial culture of Escherichia coli BL21 (DE3) transformed with XylE plasmid was added to 1 L of LB medium, cultured at 37 ° C for 4 h on a shaker at 220 rpm, and induced by adding 250 μmol/L IPTG. Incubate for 4 h at 37 ° C in a shaker at 220 rpm. BL21(DE3) cells expressing the XylE protein were collected by centrifugation, and after disrupting the cells using ultrasonic waves, the pure cell membrane fraction was separated by velocity gradient centrifugation. The XylE protein in the cell membrane is extracted by using the detergent dodecyl-β-D-maltoside (DDM), specifically, 1-2% of DDM is added to the cell membrane solution crushed by the homogenizer, Incubate for 1-2 h in a 4 ° C cold room. The XylE protein was then purified using a combination of affinity chromatography (for polyhistidine tags) and size exclusion chromatography (using a dextran 200 column). Affinity chromatography was carried out using a Ni-NTA column, and the supernatant of the membrane protein extract was added to the column. After the solution was flowed, a rinse solution (20 mmol/L imidazole, 25 mmol/L Tris buffer pH 8.0) was used. Rinsing with 150 mmol/L sodium chloride, 0.02% DDM), then eluting the protein with an eluent (250 mmol/L imidazole, 25 mmol/L Tris buffer pH 8.0, 150 mmol/L sodium chloride, 0.02% DDM) . The protein was then concentrated to 2 ml with a 50 KD concentrating tube, and subjected to molecular exclusion chromatography using a dextran 200 column. The buffer conditions were (25 mmol/L MES buffer pH 6.5, 150 mmol/L sodium chloride, 0.056%). Cymal-6).

The purified XylE protein was verified using size exclusion chromatography (see above). A single peak is shown in the resulting chromatogram, shown as a in Figure 3. In addition, the purified XylE protein was verified by SDS polyacrylamide gel electrophoresis (SDS-PAGE) using SDS-PAGE of 16% denaturing gel. The specific formula is: per ml of 60ml SDS gel: 24ml 40% acrylamide / fork acrylamide (37.5:1 by volume), 15ml 1.5M Tris buffer PH8.8, 300μl 20% SDS (m / v), 420μl 10 % ammonium persulfate, 45 μl TEMED, dilute to 60 ml with water. The resulting electrophoresis results show a single band, shown as b in Figure 3. The above results confirmed that a high purity XylE protein was obtained.

Example 2

Preparation of XylE-X0 mutant

By designing and synthesizing primers with mutation sites by molecular cloning, the tryptophan mutation Gly58Trp was introduced into the extracellular region of TM2 of wild-type XylE using conventional PCR, and the color ammonia was introduced in the extracellular region of TM8. The acid mutation Leu315Trp gave a mutant XylE (hereinafter referred to as XylE-X0).

Specifically, PCR is used to first amplify a DNA sequence from the 5' start to the mutation site in the entire sequence, and then a DNA sequence from the 3' end to the mutation site in the entire sequence is amplified, and then the two fragments are mixed. The 5' start-end forward primer and the 3' end sequence reverse primer were used to amplify the entire sequence. Among them, the primers used to introduce the Gly58Trp mutation are:

5' end primer CCCTGTTAtggTTTTGCG, 3' end primer CGCAAAAccaTAACAGGG;

The primers used to introduce the Leu315Trp mutation are:

5' end primer GATATCGCGtggTTGCAGAC, 3' end primer GTCTGCAAccaCGCGATATC.

The nucleotide residues of the mutation site are indicated in lower case letters in the primer sequence. The 5' start-end forward primer and the 3' end sequence reverse primer were the same as in Example 1. The full-length DNA sequence encoding the XylE-X0 mutant was confirmed to be the sequence of SEQ ID NO: 5, and the amino acid number of the above-mentioned mutation site corresponds to the sequence number of the amino acid in SEQ ID NO: 4. The XylE-X0 mutant has an amino acid sequence as shown in SEQ ID NO: 6. Expression and purification of the XylE-X0 mutant were carried out in the same manner as in Example 1. The purified XylE-X0 mutant protein was verified by size exclusion chromatography using the same procedure as in Example 1, and the resulting chromatogram showed a single peak, which is shown in a of Fig. 4. Further, the purified XylE-X0 mutant protein was verified by the same operation as in Example 1 using SDS-PAGE, and the obtained electrophoresis results showed a single band, which is shown in b of Fig. 4. The above results confirmed that a high purity XylE-X0 mutant protein was obtained.

Example 3

X-ray crystallography study of XylE-X0 mutant

The concentration of the purified XylE-X0 mutant protein was adjusted to about 5 mg/ml, and then crystallization was carried out by an evaporation-diffusion equilibrium crystallization method of a hanging drop. A crystal having a higher diffraction quality was obtained by using crystallization conditions of 0.1 M NaCl, 0.1 M Li ₂ SO ₄ , 0.1 M MES pH 6.5, 30% PEG 400 (v/v). After the diffraction data was collected by the Shanghai Synchrotron Radiation Center (SSRF) BL17U harness station, the atomic resolution XylE-X0 mutant structure was resolved according to the conventional molecular replacement method in the field of Non-Patent Document 9. The structure of the atomic resolution of the XylE-X0 mutant is shown in Fig. 5.

It is clear from the atomic resolution crystal structure of the XylE-X0 mutant protein that two large sterically hindered side chain amino acid residue mutations (Gly58Trp/Leu315Trp) immobilize the protein in a conformation toward the outer side of the cell membrane. The structural comparison of the substrate binding site of the XylE-X0 mutant protein with the wild-type XylE protein shown in Figure 6 further shows that the XylE-X0 mutant protein is highly consistent with the substrate binding site of the wild-type XylE protein. These results demonstrate that the introduction of a large sterically hindered side chain amino acid residue mutation does not affect the binding of the substrate, thus demonstrating that this type of mutant can be used as a prokaryotic protein model of the exo-opening GLUT conformer for screening for GLUT Binding agent.

Example 4

Transport activity of XylE and its mutants

In general, when a drug transport protein is screened for a membrane transporter, the environment in which the protein is placed is a solution environment or a lipid membrane environment. To verify that the XylE-X0 mutant was also in a fixed conformation in a drug screening environment, the inventors performed the following liposome-based transport assays and polyethylene glycol labeling experiments.

In liposome-based transport experiments, the assembly of protein liposomes was first carried out by dissolving polar lipids of Escherichia coli with chloroform and methanol solution (3:1 by volume) and blowing with nitrogen. After drying, the dried lipid was resuspended to 10-25 mg/ml using KPM solution (50 mmol/L potassium phosphate buffer pH 6.5, 2 mmol/L magnesium chloride), and repeatedly frozen and thawed by liquid nitrogen for 5 to 10 times, and then used. The 0.4 μm pore size filter was filtered back and forth 15 to 25 times, then 0.5% to 1.3% of the detergent OG was added to the solution, and incubated in a cold room at 4 ° C for half an hour, and then 0.8% to 1.5% according to the lipid concentration. (mass ratio) protein was added, and then incubated in a cold room at 4 ° C for one hour, then added to Biobead three times per 1 g of detergent to add Biobead (Avanti Polar Lipids, Inc.) to remove detergent. Each time Biobead was added, it was incubated in a cold room at 4 ° C for one hour, then repeatedly frozen and thawed by liquid nitrogen for 5 to 10 times, and then filtered back and forth 15 to 25 times with a 0.4 μm pore size filter. After ultracentrifugation, the protein lipid was removed. The plastid is resuspended to 50-120 mg/ml with the predetermined solution to replace the liposome to a sugar-free solution. In order to complete the preparation of proteoliposomes. The liposome transport experiments were then carried out as follows: All reactions were carried out at 25 °C by first adding a specific concentration of candidate binder molecules to the proteoliposome, incubating for half an hour, and then adding 1 μCi of ³ in 100 μl of KPM solution. H-labeled xylose, then add 2 μl of proteoliposome to the solution. After 30 s of reaction, the solution mixed with proteoliposome was filtered through a 0.22 μm filter, then the filter was rinsed with 2 ml of KPM solution, and finally The filter was added to 500 μl of scintillation fluid and incubated overnight before reading with a counter. The results obtained are shown in a of Figure 7. The results of SDS-PAGE of the blank liposome, the XylE-loaded liposome, and the liposome carrying the XylE-X0 mutant by the same procedure as in Example 1 are shown in b of Fig. 7.

A in Figure 7 shows that the XylE-X0 mutant introduced with two large sterically hindered side chain amino acid residue mutations completely lost the activity of the transport substrate, thus demonstrating that the mutant protein is still immobilized in the lipid membrane environment. The process of transporting the substrate cannot be completed by conformation in the outward opening. b in Figure 7 shows that the amount of protein in the wild-type XylE and XylE-X0 mutants contained in the liposomes was consistent in the transport experiments.

The polyethylene glycol labeling experiment was carried out in accordance with the method described in Xiaoming Zhou et al., Structural basis of the alternating-access mechanism in a b ile acid transporter, Nature 505, 569-573, January 2014, doi: 10.1038/nature 12811. The inventors screened for a cysteine single point mutant Ile171Cys that was only labeled with mPEG-Mal-5K in the inward open conformation using a Cys-less mutant of XylE. Thus, a single point mutation of Ile171Cys was introduced on the mutant without cysteine residue to obtain a mutant for labeling as I171C & WT. The Ile171Cys mutation and the Gly58Trp and Leu315Trp mutations were introduced on the cysteine-free residue mutant to obtain a marker mutant designated as I171C & G58W/L315W. After expression and purification of the two marker-use mutant proteins, the two mutant proteins were labeled with mPEG-Mal-5K under intact cell membrane conditions and under disrupted cell membrane conditions (sonication), respectively. The results of Western Blot after the polyethylene glycol labeling experiments of I171C & WT and I171C & G58W/L315W are shown in Figure 8.

As shown in a in Figure 8 and in the middle panel of c in Figure 8, the I171C & WT mutant protein, which does not introduce a large sterically hindered side chain amino acid residue mutation, is capable of transitioning between the outward and inward opening conformations. After sonication of the cells, the polyethylene glycol label (mPEG-Mal-5K) is able to pass the hydrophilic channel formed by the inward opening close to the cysteine site at position 171 of I171C & WT, thereby completing the labeling. As shown in b in Figure 8 and the right panel of c in Figure 8, after introduction of a large sterically hindered side chain residue mutation (tryptophan mutation), the protein is immobilized in the outward-facing conformation, whether or not ultrasound The disrupted cells, mPEG-Mal-5K, were unable to label the I171C & G58W/L315W mutant protein. This demonstrates that the G58W/L315W mutation allows the mutant protein to remain immobilized in the outward-facing conformation in a solution environment.

Example 5

Binding of XylE and its mutants to its natural substrate xylose, GLUT's natural substrate, glucose

The binding ability of the wild-type XylE protein obtained in Example 1 and the XylE-X0 mutant protein obtained in Example 2 to its natural substrate xylose was determined by isothermal calori titration experiments, and the two were determined. Binding to the natural substrate glucose of the glucose transporter GLUT. The XylE protein or its mutant at a concentration of 100 μmol/L is added to a reaction cell of an isothermal calorimeter (GE Healthcare) at a reaction temperature of 15 to 30 ° C at a concentration of 5 mmol/L to 10 mmol/L. The substrate was titrated to determine the binding force data between the two. The measurement results are shown in Fig. 9.

From the results of the isothermal calori titration experiment, both the wild-type XylE protein and the XylE-X0 mutant protein were able to bind to the natural substrate xylose and the natural substrate glucose of the glucose transporter GLUT. These results further demonstrate the feasibility of XylE or its mutants as prokaryotic homologous protein models of GLUT for screening binding agents against GLUT. Further analysis of the results of isothermal calori titration experiments, the detailed thermodynamic parameters of the binding process are shown in Table 1.

Table 1 Determination of thermodynamic parameters of the bonding process by isothermal calorimetry

The results of thermodynamic binding parameters shown in Table 1 show that the Gibbs free energy change (ΔG) of the binding process is the case when wild-type XylE binds to xylose or glucose and XylE-X0 mutant binds to xylose or glucose. Negative values confirm that the four processes are spontaneous processes. When the large sterically hindered side chain residue is introduced, the enthalpy change (ΔH) of the binding process changes from a positive value to a negative value, and the entropy decreases (ΔS). At this time, the change of Gibbs free energy is mainly caused by enthalpy change. provide. In the thermodynamic changes of transporters, the entropy change is mainly caused by conformational changes. The above results indicate that the introduction of large sterically hindered side chain residue mutations limits the conformational changes of transporters. This result is mutually confirmed with the results of Example 3 and Example 4.

The above experiments completely demonstrate that the introduction of large sterically hindered side chain residue mutations can immobilize the prokaryotic homologous protein of the glucose transporter GLUT in the outward-facing conformation without affecting its substrate binding properties, so that such mutant proteins can Used to screen for binding agents to GLUT.

Example 6

Binding of XylE and its mutants to its natural substrate, xylose, GLUT's natural substrate, glucose

The binding ability of the wild type XylE obtained in Example 1 or the XylE-X0 mutant protein obtained in Example 2 to xylose or glucose was measured using a micro thermophoresis method. Using a concentration of 300 nmol/L of XylE protein or XylE-X0 mutant, the substrate xylose was diluted 1:1 from a starting concentration of 300 μmol/L, and the substrate glucose was 1:1 from a starting concentration of 1000 μmol/L. The protein was mixed with a gradient diluted substrate by gradient dilution, after which the binding force was measured using a micro thermophoresis instrument (NanoTemper Technologies). The measurement results are shown in Fig. 10.

Figure 10 shows that the micro thermophoresis method can effectively measure the binding force of the transporter protein to the substrate molecule, and the measurement result is highly consistent with the results measured by the isothermal calorimetry experiment. It was thus demonstrated that the binding ability of the candidate molecule to the prokaryotic homologous protein of GLUT can be determined using a micro thermophoresis method, and the method of binding agent screening or drug screening can be performed by this method.

Example 7

Binding of XylE and its mutants to small molecule inhibitors of GLUT

The following experiments were performed to investigate the binding of two well-confirmed small molecule inhibitors of GLUT to wild-type XylE and XylE-X0 mutant proteins. The two small molecule inhibitors are phloretin and cytochalasin B (CCB), the molecular structure of which is shown in the following formula.

a. Liposomes-based transport experiments

The liposome-based transport assay was carried out by the same method as in Example 4, and the results of inhibition of the transport activity of the two small molecule inhibitors against wild-type XylE are shown in Fig. 11. Both small molecule inhibitors have a significant inhibitory effect on the transport activity of wild-type XylE protein.

b. Micro thermophoresis experiment

The micro thermophoresis experiment was carried out by the same method as in Example 6. The dissociation constant (K _d ) of phloretin and CCB and the wild type XylE and XylE-X0 mutants were measured by microcalorimetry, respectively, and the results are shown in Fig. 12.

The results of the binding experiments determined by microcalorimetry showed that both phloretin and CCB, which are GLUT inhibitors, can bind to wild-type XylE, confirming that the candidate molecule binds to GLUT by binding experiments with XylE. No and/or assess the binding of the candidate molecule to the GLUT. At the same time, phloretin can bind to XylE-X0, while CCB cannot bind to XylE-X0. This suggests that phloretin can bind to the outward-facing conformational isomer of the GLUT protein, whereas CCB can bind to the inward-opening conformer of the GLUT protein, a result consistent with several articles already reported, such as Tabitha E. Wood et al., A novel inhibitor of glucose uptake sensitizes cells to FAS-induced cell death, Molecular Cancer Therapeutics, 10.1158/1535-7163. MCT-08-0569, November 2008 and Khyati Kapoor et al., Mechanism of inhibition Of human glucose transporter GLUT1is conserved between cytochalasin B and phenylalanine amides, PNAS 113 (17), 4711-4716, doi: 10.1073/pnas. 1603735113 (2016).

The above results demonstrate that the method of the present disclosure can be used not only to screen for binding agents against GLUT, but also to evaluate the binding ability of the binding agent to GLUT, and also to determine the position of the binding agent acting on the GLUT, thereby further Drug design optimization provides a variety of information.

Comparative example 1

Preparation of GlcPse protein

The full-length DNA encoding the GlcPse protein was amplified using the 5' start-end forward primer: gatgcacatATGAAAGCGAACAAGTACCTG and the 3'-end reverse primer: cggatcctcgagttaTTCGGTACGCGCGCCAG, and the sequence shown by SEQ ID NO: 51 was confirmed by sequencing.

The DNA encoding the GlcPse protein was cloned into the pET15b vector (Novagen) by molecular cloning means, and the expression in the E. coli BL21 (DE3) system was the same as in Example 1 except that the IPTG induction concentration was 400 μmol/L. The GlcPse protein of the amino acid sequence shown in SEQ ID NO: 52. To purify the expressed GlcPse protein, size exclusion chromatography was carried out using the following buffer: 25 mmol/L Tris pH 8.0, 150 mmol/L NaCl, 0.02% DDM, and the rest of the procedure was the same as in Example 1. SDS-PAGE was carried out in the same manner as in Example 1.

The purified GlcPse protein was verified using size exclusion chromatography (see above). The resulting chromatogram shows a main peak between fractions 10 to 16, shown as a in Figure 13. Fractions 12 to 14 obtained by size exclusion chromatography were analyzed by SDS-PAGE, and the obtained electrophoresis results showed a single band, which is shown in b of Fig. 13. The above results confirmed that a high purity GlcPse protein was obtained.

Comparative example 2

Preparation of GlcP-6 mutant

The preparation of a mutant of GlcPse protein having Gly45Trp/Ile277Trp double tryptophan mutation (hereinafter referred to as GlcP-6 mutant) was carried out in the same manner as in Example 2. The primers used to introduce the Gly45Trp mutation are:

5' end primer GCACCACCGAAtggATTGTGGTGAGC, 3' end primer GCTCACCACAATccaTTCGGTGGTGC;

The primers used to introduce the Ile277Trp mutation are:

The 5' primer GCGGCGAGCtggTTAGGTAGC, the 3' primer GCTACCTAAccaGCTCGCCGC.

The nucleotide residues of the mutation site are indicated in lower case letters in the primer sequence. The 5' start-end forward primer and the 3'-end reverse primer were the same as in Comparative Example 1. The full-length DNA sequence of the obtained GlcP-6 mutant was confirmed to be the sequence shown in SEQ ID NO: 53, and the amino acid number of the above-mentioned mutation site corresponds to the sequence. The number of the amino acid in No. 52. The DNA encoding the GlcP-6 mutant was cloned into the pET15b vector (Novagen) by molecular cloning means, and the GlcP-6 mutant having the amino acid sequence of SEQ ID NO: 54 was expressed and purified by the same procedure as in Comparative Example 1. .

The purified GlcP-6 mutant was verified using the same size exclusion chromatography as in Comparative Example 1. The resulting chromatogram is shown in a of Figure 14. The chromatograms show that a large amount of GlcP-6 mutant protein is retained on the exclusion chromatography column and cannot be purified by size exclusion chromatography. The fractions Nos. 7 to 17 obtained by size exclusion chromatography were analyzed by SDS-PAGE, and the obtained electrophoresis results are shown in b of Fig. 14. The electrophoresis results showed that the GlcP-6 mutant had poor purification effect, low yield and obvious impurities. The above results show that the GlcP-6 mutant has poor stability in a solution environment.

Comparative example 3

Binding of GlcPse and its mutants to GLUT's natural substrate glucose

First, the binding of the GlcPse protein obtained in Comparative Example 1 and the GlcP-6 mutant obtained in Comparative Example 2 to glucose was measured by isothermal calorimetry. The operation of the isothermal calorimetric experiment was the same as in Example 5 except that the buffer was 25 mmol/L Tris pH 8.0, 150 mmol/L NaCl, 0.02% DDM, and the results are shown in Fig. 15.

Further, the binding of the GlcPse protein obtained in Comparative Example 1 and the GlcP-6 mutant obtained in Comparative Example 2 to glucose was measured by a micro thermophoresis method. The operation of the micro thermophoresis experiment was the same as in Example 6 except that the buffer was 25 mmol/L Tris pH 8.0, 150 mmol/L NaCl, 0.02% DDM, and the results are shown in Fig. 16.

As can be seen from Fig. 15 and Fig. 16, the binding of the GlcPse protein or the GlcP-6 mutant to the natural substrate glucose of GLUT could not be detected by isothermal calorimetry or microcalorimetry. These results suggest that GlcPse or its GlcP-6 mutant (Gly45Trp/Ile277Trp) is not suitable as a prokaryotic protein model of GLUT or its conformational isomer for screening for binding agents against GLUT by detecting binding of prokaryotic protein models to candidate molecules. Methods.

It should be noted that, as a homologous protein of XylE, the Gly45 and Ile277 sites of GlcPse represented by SEQ ID NO: 52 correspond to the Gly58 and Leu315 sites of XylE shown in SEQ ID NO: 4, and can be easily confirmed by those skilled in the art. Such a correspondence.

Comparative example 4

In addition to the above-mentioned GlcPse wild-type protein and GlcP-6 mutant, the inventors also attempted to express and analyze various GlcPse mutants shown in Table 2, however, it was found that these mutants did not have sufficient stability in a solution environment. It is not suitable as a prokaryotic protein model of GLUT or its conformational isomer (results not shown).

Table 2 Mutants of GlcPse

Mutant number Mutation relative to wild-type GlcPse GlcP-2 Gly45Trp Gly24Trp GlcP-3 Gly45Trp Ser27Trp GlcP-4 Gly45Trp Leu31Trp GlcP-5 Gly45Trp Leu145Trp GlcP-6 Gly45Trp Ile277Trp GlcP-7 Gly45Trp Ser280Trp GlcP-8 Gly45Trp Val281Trp GlcP-9 Gly45Trp Gly284Trp GlcP-10 Ser49Trp Gly24Trp GlcP-11 Ser49Trp Ser27Trp GlcP-12 Ser49Trp Leu31Trp GlcP-13 Ser49Trp Leu145Trp GlcP-14 Ser49Trp Ile277Trp GlcP-15 Ser49Trp Ser280Trp GlcP-16 Ser49Trp Val281Trp GlcP-17 Ser49Trp Gly284Trp GlcP-18 Leu52Trp Gly24Trp GlcP-19 Leu52Trp Ser27Trp GlcP-20 Leu52Trp Leu31Trp GlcP-21 Leu52Trp Leu145Trp GlcP-22 Leu52Trp Ile277Trp GlcP-23 Leu52Trp Ser280Trp GlcP-24 Leu52Trp Val281Trp GlcP-25 Leu52Trp Gly284Trp GlcP-26 Leu385Trp Gly24Trp GlcP-27 Leu385Trp Ser27Trp GlcP-28 Leu385Trp Leu31Trp GlcP-29 Leu385Trp Leu145Trp GlcP-30 Leu385Trp Ile277Trp GlcP-31 Leu385Trp Ser280Trp GlcP-32 Leu385Trp Val281Trp GlcP-33 Leu385Trp Gly284Trp GlcP-34 Ser388Trp Gly24Trp GlcP-35 Ser388Trp Ser27Trp

GlcP-36 Ser388Trp Leu31Trp GlcP-37 Ser388Trp Leu145Trp GlcP-38 Ser388Trp Ile277Trp GlcP-39 Ser388Trp Ser280Trp GlcP-40 Ser388Trp Val281Trp GlcP-41 Ser388Trp Gly284Trp GlcP-42 Leu389Trp Gly24Trp GlcP-43 Leu389Trp Ser27Trp GlcP-44 Leu389Trp Leu31Trp GlcP-45 Leu389Trp Leu145Trp GlcP-46 Leu389Trp Ile277Trp GlcP-47 Leu389Trp Ser280Trp GlcP-48 Leu389Trp Val281Trp GlcP-49 Leu389Trp Gly284Trp GlcP-50 Ile393Trp Gly24Trp GlcP-51 Ile393Trp Ser27Trp GlcP-52 Ile393Trp Leu31Trp GlcP-53 Ile393Trp Leu145Trp GlcP-54 Ile393Trp Ile277Trp GlcP-55 Ile393Trp Ser280Trp GlcP-56 Ile393Trp Val281Trp GlcP-57 Ile393Trp Gly284Trp

Example 8

Other XylE mutants that can be used as prokaryotic protein models of GLUT

Based on structural analysis and experimental verification, the inventors confirmed that by introducing a large sterically hindered side chain amino acid residue at other sites in the two mutated regions shown in Table 3, it is also possible to fix the conformation of the protein to an outwardly open type. effect. These XylE mutants can also be used in the methods of the present disclosure to screen for binding agents to GLUT.

Table 3 Large sterically hindered side chain amino acid residue mutation sites on XylE

The XylE mutant protein shown in Table 4 was constructed and prepared by the same method as in Example 2.

Table 4 Partial XylE mutants for screening for binding agents to GLUT

Mutant number Mutation site Amino acid sequence Nucleotide sequence XylE-X1 Gly58Trp Ala29Trp Serial number 8 Serial number 7 XylE-X2 Gly58Trp Ser32Trp Serial number 10 Serial number 9 XylE-X3 Gly58Trp Glu36Trp Serial number 12 Serial number 11 XylE-X4 Gly58Trp Thr318Trp Serial number 14 Serial number 13 XylE-X5 Gly58Trp Ile319Trp Serial number 16 Serial number 15 XylE-X6 Gly58Trp Gly322Trp Serial number 18 Serial number 17 XylE-X7 Ala62Trp Ala29Trp Serial number 20 Serial number 19 XylE-X8 Ala62Trp Ser32Trp Serial number 22 Serial number 21 XylE-X9 Ala62Trp Glu36Trp Serial number 24 Serial number 23 XylE-X10 Ala62Trp Leu176Trp Serial number 26 Serial number 25 XylE-X11 Ala62Trp Leu315Trp Serial number 28 Serial number 27 XylE-X12 Ala62Trp Thr318Trp Serial number 30 Serial number 29 XylE-X13 Ala62Trp Ile319Trp Serial number 32 Serial number 31 XylE-X14 Ala62Trp Gly322Trp Serial number 34 Serial number 33 XylE-X15 Leu65Trp Ala29Trp Serial number 36 Serial number 35 XylE-X16 Leu65Trp Ser32Trp Serial number 38 Serial number 37 XylE-X17 Leu65Trp Glu36Trp Serial number 40 Serial number 39 XylE-X18 Leu65Trp Leu176Trp Serial number 42 Serial number 41 XylE-X19 Leu65Trp Leu315Trp Serial number 44 Serial number 43 XylE-X20 Leu65Trp Thr318Trp Serial number 46 Serial number 45 XylE-X21 Leu65Trp Ile319Trp Serial number 48 Serial number 47 XylE-X22 Leu65Trp Gly322Trp Serial number 50 Serial number 49 XylE-X23 Gly58Trp Leu176Trp Serial number 68 Serial number 67

The binding ability of the XylE mutant protein in Table 4 to the GLUT inhibitor phloretin was determined by the same microcalorimetry as in Example 6, and the results are shown in Figures 17A-17C. The binding experiments of Figures 17A-17C show that the XylE mutants with large sterically hindered side chain amino acid residue mutations prepared by combining the mutation sites of Table 1 and the mutation region 2 shown in Table 3 are capable of The GLUT inhibitor binds to a method of screening a binding agent for GLUT of the present disclosure.

The above experimental results also suggest that: 1. at least one site selected from the mutated region 1 selected from Table 3 and at least one site selected from the mutated region 2 has a large sterically hindered side chain amino acid residue mutation. The XylE mutants all achieve the effect of immobilizing the conformation of the protein to the outwardly open type and are used in the disclosed methods of screening for binding agents to GLUT. 2. For any combination of mutation sites in 1 above, any large sterically hindered side chain residue mutations (including but not limited to tryptophan, tyrosine, phenylalanine, lysine, arginine, glutamine Acid, glutamine, aspartic acid, asparagine can achieve the same effect.

Example 9

Screening for binding agents for GLUT

Wild-type XylE was used to simulate GLUT, and XylE-X0 mutant was used to simulate the outward-opening GLUT conformer, and small molecule compounds were screened. Test library. The test library includes 25 small molecules containing molecules known to bind to GLUT. This library was used to test the effectiveness of the screening methods of the present disclosure for screening binding agents for GLUT.

The binding ability of the small molecule in the library to the wild-type XylE or XylE-X0 mutant was determined by the same microcalorimetry as in Example 6. Two of the screens were shown to show both binding to wild-type XylE and also to XylE-. The small molecule compounds bound by the X0 mutant are phloridzin and Fasentin, respectively. The structure of the two is shown in the following equation.

The results of the micro thermophoresis experiment in which phlorizin and farnestin obtained in the screening were combined with XylE or XylE-X0 mutants are shown in a of Fig. 18. A in Figure 18 shows that both phlorizin and farein can bind to wild-type XylE and XylE-X0 mutant proteins.

The results of the micro thermophoresis experiment in which the D-ribose obtained in the screening was combined with the XylE or XylE-X0 mutant are shown in b in Fig. 18. b in Figure 18 shows that no binding of D-ribose to the prokaryotic proteins of the present disclosure was detected.

Since it is known in the art that phlorizin and farein can bind to GLUT, and D-ribose cannot bind to GLUT, the above experimental results confirm that the screening method of the present disclosure using the prokaryotic protein model of the present disclosure can successfully screen for GLUT. Binding agent. In addition, since phlorizin and farein can bind to both XylE and XylE-X0 mutants which mimic the outward-opening GLUT isomer, according to the method of the present disclosure, it can be judged that the two small-molecule compounds can Binds to the outwardly open GLUT conformer.

The effect of phlorizin and farnesine on the substrate transport activity of XylE was examined by the liposome transport assay using the same procedure as in Example 4. The results are shown in Fig. 19. Figure 19 shows that both phlorizin and farsentin are capable of inhibiting the transport activity of XylE as a prokaryotic protein model of GLUT, which is consistent with the results of known phlorizin and farnestin as GLUT inhibitors.

The above is only the specific embodiment of the present disclosure, but the scope of the present disclosure is not limited thereto, and any person skilled in the art can easily think of changes or substitutions within the technical scope of the disclosure. It should be covered within the scope of protection of the present disclosure. Therefore, the scope of protection of the present disclosure should be determined by the scope of the claims.

Practicality

Using the prokaryotic protein model and method of the GLUT of the present disclosure, a large amount of information for the drug screening process can be obtained as follows: 1. Inhibition of the transport activity of the drug molecule to the prokaryotic protein model of GLUT can be performed to evaluate whether the molecule can inhibit the GLUT protein. 2. For a molecule that can inhibit the transport activity of a prokaryotic protein model of GLUT, the dissociation constant obtained by binding assay can be used to evaluate the binding strength of the molecule to the GLUT protein; 3. By comparing the drug molecule with the prokaryotic of the GLUT The protein model and the binding force of the mutant prokaryotic protein that mimics the specific conformer of the GLUT can be judged whether the molecule binds to the GLUT in the outward-facing conformation or to the GLUT in the inward-facing conformation.

Claims (50)

A method of screening a binding agent for GLUT, the method comprising the steps of 1) and/or 2) below, and the step comprising 3): 1) a step of detecting binding of a candidate molecule to a first prokaryotic protein, 2) a step of detecting the binding of the candidate molecule to the second prokaryotic protein, 3) determining the binding of the candidate molecule to the GLUT, thereby determining whether the candidate molecule is a binding agent for the GLUT; Wherein the first prokaryotic protein is a prokaryotic-derived homologous protein of GLUT, the first prokaryotic protein capable of binding to glucose, the GLUT being selected from the group consisting of GLUT1, GLUT2, GLUT3, GLUT4, GLUT5, GLUT6, GLUT7, GLUT8, GLUT9 a group consisting of GLUT10, GLUT11, GLUT12, GLUT13, GLUT14, and other GLUT subtypes, and the first prokaryotic protein has more than 35% sequence similarity to the GLUT; The second prokaryotic protein is a protein comprising a sequence having a large sterically hindered side chain amino acid residue mutation in an amino acid sequence of the first prokaryotic protein, which mimics a conformational isomer of GLUT; The large sterically hindered side chain amino acid is selected from the group consisting of tryptophan, tyrosine, phenylalanine, lysine, arginine, glutamic acid, glutamine, aspartic acid, and asparagine. group. The method according to claim 1, wherein in step 3), the combination of the candidate molecule and the GLUT is determined as follows: a), b) and/or c): a) if the binding of the candidate molecule to the first prokaryotic protein is detected in step 1), it is determined that the candidate molecule is capable of binding to the GLUT; b) if the binding of the candidate molecule to the second prokaryotic protein is detected in step 2), determining that the candidate molecule is capable of binding to the conformational isomer of the GLUT mimicked by the second prokaryotic protein; c) if the binding of the candidate molecule to the first prokaryotic protein is detected in step 1), and the binding of the candidate molecule to the second prokaryotic protein is not detected in step 2), then the candidate molecule is determined to be different from said The conformational isomer of the GLUT of the conformational isomer of the two prokaryotic proteins. The method of claim 1 or 2, wherein the second prokaryotic protein mimics an outward open-ended conformer of GLUT. The method according to claim 3, wherein in step 3), the combination of the candidate molecule and the GLUT is determined as follows: a), b) and/or c): a) if the binding of the candidate molecule to the first prokaryotic protein is detected in step 1), it is determined that the candidate molecule is capable of binding to the GLUT; b) if the binding of the candidate molecule to the second prokaryotic protein is detected in step 2), determining that the candidate molecule is capable of binding to the outward open conformational conformation of the GLUT; c) if the binding of the candidate molecule to the first prokaryotic protein is detected in step 1), and the binding of the candidate molecule to the second prokaryotic protein is not detected in step 2), then the candidate molecule can be determined to be inward-open with the GLUT Conformational isomers are combined. The method according to any one of claims 1 to 4, wherein the detecting is carried out by microcalorimetry and/or isothermal calorimetry. The method according to any one of claims 1 to 5, wherein the method further comprises the step of detecting the effect of the candidate molecule on the substrate transport activity of the first prokaryotic protein by a liposome transport assay. The method according to any one of claims 1 to 6, wherein the first prokaryotic protein has a sequence identity with XylE of 80% or more; preferably, the first prokaryotic protein is derived from Escherichia coli. The method according to any one of claims 1 to 7, wherein the first prokaryotic protein is a protein comprising an amino acid sequence of (s1), (s2) or (s3): (s1) an amino acid sequence of SEQ ID NO: (s2) a sequence in which one or more amino acids are deleted, substituted and/or added in the amino acid sequence shown in SEQ ID NO: (s3) A sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4. The method according to any one of claims 1 to 8, wherein said second prokaryotic protein is contained in an amino acid sequence of said first prokaryotic protein A protein having more than two large sterically hindered sequences in which side chain amino acid residues are mutated. The method according to claim 9, wherein at least one of the mutations of the two or more large sterically hindered side chain amino acid residues is located in the region from amino acid residues 52 to 68 of SEQ ID NO: At least one other mutation is located in a region selected from amino acid residues 25 to 40 of SEQ ID NO: 4, a region from amino acid position 172 to position 190, and amino acid residues from position 311 to position 326 The area consists of the group's area. The method according to claim 9 or 10, wherein at least one of the mutations of the two or more large sterically hindered side chain amino acid residues is at position 58 which is selected from the amino acid sequence shown by SEQ ID NO: The site of the group consisting of 62 and 65, at least one other mutation is located at the 29th, 32nd, 36th, 176th, 315th, and 315th positions of the amino acid sequence shown by SEQ ID NO: 4. The locus of the group consisting of 318, 319, and 322. The method according to any one of claims 9 to 11, wherein the two or more large sterically hindered side chain amino acid residue mutations are included in at least one of the following (a) to (x) The resulting mutation: (a) 58th and 315th of SEQ ID NO: 4; (b) 58th and 29th of SEQ ID NO: 4; (c) 58th and 32nd of SEQ ID NO: 4; (d) 58th and 36th of SEQ ID NO: 4; (e) 58th and 176th of SEQ ID NO: 4; (f) 58th and 319th of SEQ ID NO: 4; (g) 58th and 322th of SEQ ID NO: 4; (h) 62nd and 29th of SEQ ID NO: 4; (i) 62nd and 32nd of serial number 4; (j) 62nd and 36th of serial number 4; (k) 62nd and 176th of SEQ ID NO: 4; (l) 62nd and 315th of serial number 4; (m) 62nd and 318th of serial number 4; (n) 62nd and 319th of SEQ ID NO: 4; (o) 62nd and 322th of serial number 4; (p) 65th and 29th of SEQ ID NO: 4; (q) the 65th and 32nd of the serial number 4; (r) 65th and 36th of SEQ ID NO: 4; (s) 65th and 176th of SEQ ID NO: 4; (t) the 65th and 315th of the serial number 4; (u) 65th and 318th of SEQ ID NO: 4; (v) the 65th and 319th of the serial number 4; (w) the 65th and 322th of the serial number 4; (x) 58th and 318th of SEQ ID NO: 4. The method according to any one of claims 9 to 12, wherein the second prokaryotic protein is at least one of the following groups (a) to (x) which is produced in the amino acid sequence shown in SEQ ID NO: Residue-mutated sequence of protein: (a) in the amino acid sequence shown in SEQ ID NO: 4, the 58th glycine is substituted with tryptophan and the 315th leucine is replaced with tryptophan; (b) in the amino acid sequence shown in SEQ ID NO: 4, the 58th glycine is substituted with tryptophan and the 29th alanine is replaced with tryptophan; (c) in the amino acid sequence shown in SEQ ID NO: 4, the 58th glycine is substituted with tryptophan and the 32th serine is replaced with tryptophan; (d) in the amino acid sequence shown in SEQ ID NO: 4, the glycine at position 58 is replaced with tryptophan, and the glutamic acid at position 36 is replaced with tryptophan; (e) in the amino acid sequence shown in SEQ ID NO: 4, the 58th glycine is substituted with tryptophan, and the 176th leucine is replaced with tryptophan; (f) in the amino acid sequence shown in SEQ ID NO: 4, the 58th glycine is substituted with tryptophan, and the 319th isoleucine is substituted with tryptophan; (g) in the amino acid sequence shown in SEQ ID NO: 4, the glycine at position 58 is replaced with tryptophan, and the glycine at position 322 is replaced with tryptophan; (h) in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan, and the alanine at position 29 is replaced with tryptophan; (i) in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan, and the serine at position 32 is replaced with tryptophan; (j) in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan, and the glutamic acid at position 36 is replaced with tryptophan; (k) in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan, and the leucine at position 176 is replaced with tryptophan; (l) in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan, and the leucine at position 315 is replaced with tryptophan; (m) in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan, and the threonine at position 318 is replaced with tryptophan; (n) in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan, and the isoleucine at position 319 is replaced with tryptophan; (o) in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan, and the glycine at position 322 is replaced with tryptophan; (p) in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan, and the alanine at position 29 is replaced with tryptophan; (q) in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan, and the serine at position 32 is replaced with tryptophan; (r) in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan, and the glutamic acid at position 36 is replaced with tryptophan; (s) in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan, and the leucine at position 176 is replaced with tryptophan; (t) in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan, and the leucine at position 315 is replaced with tryptophan; (u) in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan, and the threonine at position 318 is replaced with tryptophan; (v) in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan, and the isoleucine at position 319 is replaced with tryptophan; (w) in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan, and the glycine at position 322 is replaced with tryptophan; (x) In the amino acid sequence shown in SEQ ID NO: 4, the 58th glycine was replaced with tryptophan and the 318th threonine was replaced with tryptophan. The method according to any one of claims 1 to 13, wherein the binding agent inhibits the transport activity of GLUT; preferably, the binding agent is used for cancer treatment. a kit for screening a binding agent against GLUT, the kit comprising a first prokaryotic protein and a second prokaryotic protein; Wherein the first prokaryotic protein is a prokaryotic-derived homologous protein of GLUT, the first prokaryotic protein capable of binding to glucose, the GLUT being selected from the group consisting of GLUT1, GLUT2, GLUT3, GLUT4, GLUT5, GLUT6, GLUT7, GLUT8, GLUT9 a group consisting of GLUT10, GLUT11, GLUT12, GLUT13, GLUT14, and other GLUT subtypes, and the first prokaryotic protein has more than 35% sequence similarity to the GLUT; The second prokaryotic protein is a protein comprising a sequence having a large sterically hindered side chain amino acid residue mutation in an amino acid sequence of the first prokaryotic protein, which mimics a conformational isomer of GLUT; The large sterically hindered side chain amino acid is selected from the group consisting of tryptophan, tyrosine, phenylalanine, lysine, arginine, glutamic acid, glutamine, aspartic acid, and asparagine. group. The kit according to claim 15, wherein said second prokaryotic protein mimics an outward open-ended conformer of GLUT. The kit according to claim 15 or 16, wherein the kit further comprises a liposome of the first prokaryotic protein. The kit according to any one of claims 15 to 17, wherein the first prokaryotic protein has a sequence identity with XylE of 80% or more; preferably, the first prokaryotic protein is derived from Escherichia coli. The kit according to any one of claims 15 to 18, wherein the first prokaryotic protein is a protein comprising an amino acid sequence of (s1), (s2) or (s3): (s1) an amino acid sequence of SEQ ID NO: (s2) a sequence in which one or more amino acids are deleted, substituted and/or added in the amino acid sequence shown in SEQ ID NO: (s3) A sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4. The kit according to any one of claims 15 to 19, wherein the second prokaryotic protein is a mutation comprising two or more large sterically hindered side chain amino acid residues in an amino acid sequence of the first prokaryotic protein. The sequence of the protein. The kit according to claim 20, wherein at least one of the mutations of the two or more large sterically hindered side chain amino acid residues is located in the region from amino acid residues 52 to 68 of SEQ ID NO: At least one other mutation is located in a region selected from amino acid residues 25 to 40 of SEQ ID NO: 4, a region from amino acids 172 to 190, and amino acid residues from 311 to 326 The area of the group consisting of the base area. The kit according to claim 20 or 21, wherein at least one of the mutations of the two or more large sterically hindered side chain amino acid residues is at 58th position selected from the amino acid sequence shown by SEQ ID NO: The site of the group consisting of the 62nd and the 65th, at least one other mutation is located at the 29th, 32nd, 36th, 176th, and 315th positions of the amino acid sequence shown by SEQ ID NO: 4. The locus of the group consisting of 318th, 319th, and 322th. The kit according to any one of claims 20 to 22, wherein the two or more large sterically hindered side chain amino acid residue mutations are included in at least one of the following (a) to (x) The mutation produced on: (a) 58th and 315th of SEQ ID NO: 4; (b) 58th and 29th of SEQ ID NO: 4; (c) 58th and 32nd of SEQ ID NO: 4; (d) 58th and 36th of SEQ ID NO: 4; (e) 58th and 176th of SEQ ID NO: 4; (f) 58th and 319th of SEQ ID NO: 4; (g) 58th and 322th of SEQ ID NO: 4; (h) 62nd and 29th of SEQ ID NO: 4; (i) 62nd and 32nd of serial number 4; (j) 62nd and 36th of serial number 4; (k) 62nd and 176th of SEQ ID NO: 4; (l) 62nd and 315th of serial number 4; (m) 62nd and 318th of serial number 4; (n) 62nd and 319th of SEQ ID NO: 4; (o) 62nd and 322th of serial number 4; (p) 65th and 29th of SEQ ID NO: 4; (q) the 65th and 32nd of the serial number 4; (r) 65th and 36th of SEQ ID NO: 4; (s) 65th and 176th of SEQ ID NO: 4; (t) the 65th and 315th of the serial number 4; (u) 65th and 318th of SEQ ID NO: 4; (v) the 65th and 319th of the serial number 4; (w) the 65th and 322th of the serial number 4; (x) 58th and 318th of SEQ ID NO: 4. The kit according to any one of claims 20 to 23, wherein the second prokaryotic protein is at least one of the following (a) to (x) generated in the amino acid sequence shown in SEQ ID NO: A protein with a sequence of a mutated amino acid residue: (a) in the amino acid sequence shown in SEQ ID NO: 4, the 58th glycine is substituted with tryptophan and the 315th leucine is replaced with tryptophan; (b) in the amino acid sequence shown in SEQ ID NO: 4, the 58th glycine is substituted with tryptophan and the 29th alanine is replaced with tryptophan; (c) in the amino acid sequence shown in SEQ ID NO: 4, the 58th glycine is substituted with tryptophan and the 32th serine is replaced with tryptophan; (d) in the amino acid sequence shown in SEQ ID NO: 4, the glycine at position 58 is replaced with tryptophan, and the glutamic acid at position 36 is replaced with tryptophan; (e) in the amino acid sequence shown in SEQ ID NO: 4, the 58th glycine is substituted with tryptophan, and the 176th leucine is replaced with tryptophan; (f) in the amino acid sequence shown in SEQ ID NO: 4, the 58th glycine is substituted with tryptophan, and the 319th isoleucine is substituted with tryptophan; (g) in the amino acid sequence shown in SEQ ID NO: 4, the glycine at position 58 is replaced with tryptophan, and the glycine at position 322 is replaced with tryptophan; (h) in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan, and the alanine at position 29 is replaced with tryptophan; (i) in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan, and the serine at position 32 is replaced with tryptophan; (j) in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan, and the glutamic acid at position 36 is replaced with tryptophan; (k) in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan, and the leucine at position 176 is replaced with tryptophan; (l) in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan, and the leucine at position 315 is replaced with tryptophan; (m) in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan, and the threonine at position 318 is replaced with tryptophan; (n) in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan, and the isoleucine at position 319 is replaced with tryptophan; (o) in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan, and the glycine at position 322 is replaced with tryptophan; (p) in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan, and the alanine at position 29 is replaced with tryptophan; (q) in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan, and the serine at position 32 is replaced with tryptophan; (r) in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan, and the glutamic acid at position 36 is replaced with tryptophan; (s) in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan, and the leucine at position 176 is replaced with tryptophan; (t) in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan, and the leucine at position 315 is replaced with tryptophan; (u) in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan, and the threonine at position 318 is replaced with tryptophan; (v) in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan, and the isoleucine at position 319 is replaced with tryptophan; (w) in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan, and the glycine at position 322 is replaced with tryptophan; (x) In the amino acid sequence shown in SEQ ID NO: 4, the 58th glycine was replaced with tryptophan and the 318th threonine was replaced with tryptophan. The kit according to any one of claims 15 to 24, wherein the binding agent inhibits the transport activity of GLUT; preferably, the binding agent is used for cancer treatment. An isolated prokaryotic protein mimicking GLUT, which is a prokaryotic-derived homologous protein of GLUT, wherein the prokaryotic protein is capable of binding glucose, The GLUT is selected from the group consisting of GLUT1, GLUT2, GLUT3, GLUT4, GLUT5, GLUT6, GLUT7, GLUT8, GLUT9, GLUT10, GLUT11, GLUT12, GLUT13, GLUT14, and other GLUT subtypes, and the prokaryotic protein and the GLUT Has a sequence similarity of more than 35%. The prokaryotic protein according to claim 26, which has a sequence identity with XylE of 80% or more; preferably, the prokaryotic protein is derived from Escherichia coli. The prokaryotic protein according to claim 26 or 27, which is a protein comprising the amino acid sequence of (s1), (s2) or (s3): (s1) an amino acid sequence of SEQ ID NO: (s2) a sequence in which one or more amino acids are deleted, substituted and/or added in the amino acid sequence shown in SEQ ID NO: (s3) A sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4. An isolated prokaryotic protein that mimics the conformational isomer of GLUT, which is a protein comprising a sequence having a large sterically hindered side chain amino acid residue mutation in the amino acid sequence of a prokaryotic-derived homologous protein of GLUT, Prokaryotic-derived homologous proteins of GLUT are capable of binding to glucose, Wherein the GLUT is selected from the group consisting of GLUT1, GLUT2, GLUT3, GLUT4, GLUT5, GLUT6, GLUT7, GLUT8, GLUT9, GLUT10, GLUT11, GLUT12, GLUT13, GLUT14, and other GLUT subtypes, and the prokaryotic source of the GLUT a homologous protein having more than 35% sequence similarity to the GLUT, and The large sterically hindered side chain amino acid is selected from the group consisting of tryptophan, tyrosine, phenylalanine, lysine, arginine, glutamic acid, glutamine, aspartic acid, and asparagine. group. A prokaryotic protein according to claim 29 which mimics the outward-facing conformational isomer of GLUT. The prokaryotic protein according to claim 29 or 30, wherein the prokaryotic-derived homologous protein of the GLUT has a sequence identity of more than 80% with XylE; preferably, the prokaryotic source of the prokaryotic source of the GLUT In E. coli. The prokaryotic protein according to any one of claims 29 to 31, which has two or more large sterically hindered side chain amino acid residues in an amino acid sequence of a protein comprising (p1), (p2) or (p3) Basis-mutated sequence of proteins: (p1) a protein having the amino acid sequence of SEQ ID NO: 4; (p2) a protein having the sequence of deleting, replacing and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO: (p3) A protein having a sequence having a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 4. The prokaryotic protein according to claim 32, wherein at least one of the mutations of the two or more large sterically hindered side chain amino acid residues is located in the region from amino acid residues 52 to 68 of SEQ ID NO: At least one other mutation is located in a region selected from amino acid residues 25 to 40 of SEQ ID NO: 4, a region from amino acids 172 to 190, and amino acid residues from 311 to 326 The area of the group consisting of the base area. The prokaryotic protein according to claim 32 or 33, wherein at least one of the mutations of the two or more large sterically hindered side chain amino acid residues is at 58th position selected from the amino acid sequence shown by SEQ ID NO: The site of the group consisting of the 62nd and the 65th, at least one other mutation is located at the 29th, 32nd, 36th, 176th, and 315th positions of the amino acid sequence shown by SEQ ID NO: 4. The locus of the group consisting of 318th, 319th, and 322th. The prokaryotic protein according to any one of claims 32 to 34, wherein the two or more large sterically hindered side chain amino acid residue mutations are included in at least one of the following (a) to (x) The mutation produced on: (a) 58th and 315th of SEQ ID NO: 4; (b) 58th and 29th of SEQ ID NO: 4; (c) 58th and 32nd of SEQ ID NO: 4; (d) 58th and 36th of SEQ ID NO: 4; (e) 58th and 176th of SEQ ID NO: 4; (f) 58th and 319th of SEQ ID NO: 4; (g) 58th and 322th of SEQ ID NO: 4; (h) 62nd and 29th of SEQ ID NO: 4; (i) 62nd and 32nd of serial number 4; (j) 62nd and 36th of serial number 4; (k) 62nd and 176th of SEQ ID NO: 4; (l) 62nd and 315th of serial number 4; (m) 62nd and 318th of serial number 4; (n) 62nd and 319th of SEQ ID NO: 4; (o) 62nd and 322th of serial number 4; (p) 65th and 29th of SEQ ID NO: 4; (q) the 65th and 32nd of the serial number 4; (r) 65th and 36th of SEQ ID NO: 4; (s) 65th and 176th of SEQ ID NO: 4; (t) the 65th and 315th of the serial number 4; (u) 65th and 318th of SEQ ID NO: 4; (v) the 65th and 319th of the serial number 4; (w) the 65th and 322th of the serial number 4; (x) 58th and 318th of SEQ ID NO: 4. The prokaryotic protein according to any one of claims 32 to 35, which is a sequence comprising the amino acid sequence shown in SEQ ID NO: 4 which produces a mutation of at least one of the following (a) to (x) Protein: (a) in the amino acid sequence shown in SEQ ID NO: 4, the 58th glycine is substituted with tryptophan and the 315th leucine is replaced with tryptophan; (b) in the amino acid sequence shown in SEQ ID NO: 4, the 58th glycine is substituted with tryptophan and the 29th alanine is replaced with tryptophan; (c) in the amino acid sequence shown in SEQ ID NO: 4, the 58th glycine is substituted with tryptophan and the 32th serine is replaced with tryptophan; (d) in the amino acid sequence shown in SEQ ID NO: 4, the glycine at position 58 is replaced with tryptophan, and the glutamic acid at position 36 is replaced with tryptophan; (e) in the amino acid sequence shown in SEQ ID NO: 4, the 58th glycine is substituted with tryptophan, and the 176th leucine is replaced with tryptophan; (f) in the amino acid sequence shown in SEQ ID NO: 4, the 58th glycine is substituted with tryptophan, and the 319th isoleucine is substituted with tryptophan; (g) in the amino acid sequence shown in SEQ ID NO: 4, the glycine at position 58 is replaced with tryptophan, and the glycine at position 322 is replaced with tryptophan; (h) in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan, and the alanine at position 29 is replaced with tryptophan; (i) in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan, and the serine at position 32 is replaced with tryptophan; (j) in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan, and the glutamic acid at position 36 is replaced with tryptophan; (k) in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan, and the leucine at position 176 is replaced with tryptophan; (l) in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan, and the leucine at position 315 is replaced with tryptophan; (m) in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan, and the threonine at position 318 is replaced with tryptophan; (n) In the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan, and the isoleucine at position 319 is replaced with color ammonia. acid; (o) in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan, and the glycine at position 322 is replaced with tryptophan; (p) in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan, and the alanine at position 29 is replaced with tryptophan; (q) in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan, and the serine at position 32 is replaced with tryptophan; (r) in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan, and the glutamic acid at position 36 is replaced with tryptophan; (s) in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan, and the leucine at position 176 is replaced with tryptophan; (t) in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan, and the leucine at position 315 is replaced with tryptophan; (u) in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan, and the threonine at position 318 is replaced with tryptophan; (v) in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan, and the isoleucine at position 319 is replaced with tryptophan; (w) in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan, and the glycine at position 322 is replaced with tryptophan; (x) In the amino acid sequence shown in SEQ ID NO: 4, the 58th glycine was replaced with tryptophan and the 318th threonine was replaced with tryptophan. A polynucleotide encoding the prokaryotic protein of any one of claims 26 to 36. An expression vector comprising the polynucleotide of claim 37. A transformant obtained by transforming a host with the expression vector of claim 38. The transformant according to claim 39, wherein the host is Escherichia coli. A method for producing a prokaryotic protein which mimics a conformational isomer of GLUT or GLUT, which comprises the polynucleotide of claim 37, the expression vector of claim 38 or the transformant of claim 39 or 40. The prokaryotic protein is obtained. A method for immobilizing the conformation of a prokaryotic-derived homologous protein of GLUT, characterized in that the same is fixed by introducing a large sterically hindered side chain amino acid residue mutation into a homologous protein derived from a prokaryote of the GLUT A conformation of a source protein, wherein the homologous protein is capable of binding to glucose, the GLUT being selected from the group consisting of GLUT1, GLUT2, GLUT3, GLUT4, GLUT5, GLUT6, GLUT7, GLUT8, GLUT9, GLUT10, GLUT11, GLUT12, GLUT13, GLUT14, and other GLUTs a group consisting of subtypes, and the homologous protein has more than 35% sequence similarity to the GLUT, The large sterically hindered side chain amino acid is selected from the group consisting of tryptophan, tyrosine, phenylalanine, lysine, arginine, glutamic acid, glutamine, aspartic acid, and asparagine. group. 40. The method of claim 42, wherein the homologous protein is immobilized in an outward open conformation. The method according to claim 42 or 43, wherein the homologous protein has a sequence identity with XylE of 80% or more; preferably, the homologous protein is derived from Escherichia coli. The method according to any one of claims 42 to 44, wherein the homologous protein is a protein comprising an amino acid sequence of (s1), (s2) or (s3): (s1) an amino acid sequence of SEQ ID NO: (s2) a sequence in which one or more amino acids are deleted, substituted and/or added in the amino acid sequence shown in SEQ ID NO: (s3) A sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4. The method according to any one of claims 42 to 45, wherein two or more large sterically hindered side chain amino acid residue mutations are introduced in the amino acid sequence of the homologous protein. The method according to claim 46, wherein at least one of said two or more large sterically hindered side chain amino acid residue mutations is located in a region of SEQ ID NO: 4 from amino acid residues 52 to 68, At least one other mutation is located in a region selected from amino acid residues 25 to 40 of SEQ ID NO: 4, a region from amino acid position 172 to position 190, and amino acid residues from position 311 to position 326 The area consists of the group's area. The method according to claim 46 or 47, wherein at least one of the mutations of the two or more large sterically hindered side chain amino acid residues is at position 58 selected from the amino acid sequence shown by SEQ ID NO: The site of the group consisting of 62 and 65, at least one other mutation is located at the 29th, 32nd, 36th, 176th, 315th, and 315th positions of the amino acid sequence shown by SEQ ID NO: 4. The locus of the group consisting of 318, 319, and 322. The method according to any one of claims 46 to 48, wherein the two or more large sterically hindered side chain amino acid residue mutations are included in at least one of the following (a) to (x) The resulting mutation: (a) 58th and 315th of SEQ ID NO: 4; (b) 58th and 29th of SEQ ID NO: 4; (c) 58th and 32nd of SEQ ID NO: 4; (d) 58th and 36th of SEQ ID NO: 4; (e) 58th and 176th of SEQ ID NO: 4; (f) 58th and 319th of SEQ ID NO: 4; (g) 58th and 322th of SEQ ID NO: 4; (h) 62nd and 29th of SEQ ID NO: 4; (i) 62nd and 32nd of serial number 4; (j) 62nd and 36th of serial number 4; (k) 62nd and 176th of SEQ ID NO: 4; (l) 62nd and 315th of serial number 4; (m) 62nd and 318th of serial number 4; (n) 62nd and 319th of SEQ ID NO: 4; (o) 62nd and 322th of serial number 4; (p) 65th and 29th of SEQ ID NO: 4; (q) the 65th and 32nd of the serial number 4; (r) 65th and 36th of SEQ ID NO: 4; (s) 65th and 176th of SEQ ID NO: 4; (t) the 65th and 315th of the serial number 4; (u) 65th and 318th of SEQ ID NO: 4; (v) the 65th and 319th of the serial number 4; (w) the 65th and 322th of the serial number 4; (x) 58th and 318th of SEQ ID NO: 4. The method according to any one of claims 46 to 49, wherein the mutation of the two or more large sterically hindered side chain amino acid residues comprises mutation of at least one of the following (a) to (x) : (a) in the amino acid sequence shown in SEQ ID NO: 4, the 58th glycine is substituted with tryptophan and the 315th leucine is replaced with tryptophan; (b) in the amino acid sequence shown in SEQ ID NO: 4, the 58th glycine is substituted with tryptophan and the 29th alanine is replaced with tryptophan; (c) in the amino acid sequence shown in SEQ ID NO: 4, the 58th glycine is substituted with tryptophan and the 32th serine is replaced with tryptophan; (d) in the amino acid sequence shown in SEQ ID NO: 4, the glycine at position 58 is replaced with tryptophan, and the glutamic acid at position 36 is replaced with tryptophan; (e) in the amino acid sequence shown in SEQ ID NO: 4, the 58th glycine is substituted with tryptophan, and the 176th leucine is replaced with tryptophan; (f) In the amino acid sequence shown in SEQ ID NO: 4, the 58th glycine is replaced with tryptophan, and the 319th isoleucine is substituted with color ammonia. acid; (g) in the amino acid sequence shown in SEQ ID NO: 4, the glycine at position 58 is replaced with tryptophan, and the glycine at position 322 is replaced with tryptophan; (h) in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan, and the alanine at position 29 is replaced with tryptophan; (i) in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan, and the serine at position 32 is replaced with tryptophan; (j) in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan, and the glutamic acid at position 36 is replaced with tryptophan; (k) in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan, and the leucine at position 176 is replaced with tryptophan; (l) in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan, and the leucine at position 315 is replaced with tryptophan; (m) in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan, and the threonine at position 318 is replaced with tryptophan; (n) in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan, and the isoleucine at position 319 is replaced with tryptophan; (o) in the amino acid sequence shown in SEQ ID NO: 4, the alanine at position 62 is replaced with tryptophan, and the glycine at position 322 is replaced with tryptophan; (p) in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan, and the alanine at position 29 is replaced with tryptophan; (q) in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan, and the serine at position 32 is replaced with tryptophan; (r) in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan, and the glutamic acid at position 36 is replaced with tryptophan; (s) in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan, and the leucine at position 176 is replaced with tryptophan; (t) in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan, and the leucine at position 315 is replaced with tryptophan; (u) in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan, and the threonine at position 318 is replaced with tryptophan; (v) in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan, and the isoleucine at position 319 is replaced with tryptophan; (w) in the amino acid sequence shown in SEQ ID NO: 4, the leucine at position 65 is replaced with tryptophan, and the glycine at position 322 is replaced with tryptophan; (x) In the amino acid sequence shown in SEQ ID NO: 4, the 58th glycine was replaced with tryptophan and the 318th threonine was replaced with tryptophan.

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