WO2023234513A1 - Capteur pour détecter la glutamine à l'aide d'une protéine de liaison à la glutamine scindée et son utilisation - Google Patents

Capteur pour détecter la glutamine à l'aide d'une protéine de liaison à la glutamine scindée et son utilisation Download PDF

Info

Publication number
WO2023234513A1
WO2023234513A1 PCT/KR2023/001429 KR2023001429W WO2023234513A1 WO 2023234513 A1 WO2023234513 A1 WO 2023234513A1 KR 2023001429 W KR2023001429 W KR 2023001429W WO 2023234513 A1 WO2023234513 A1 WO 2023234513A1
Authority
WO
WIPO (PCT)
Prior art keywords
glutamine
domain
protein
fluorescent protein
seq
Prior art date
Application number
PCT/KR2023/001429
Other languages
English (en)
Korean (ko)
Inventor
서문형
박근완
정제형
이욱빈
김호연
이제훈
Original Assignee
한국과학기술연구원
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 한국과학기술연구원 filed Critical 한국과학기술연구원
Publication of WO2023234513A1 publication Critical patent/WO2023234513A1/fr

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6806—Determination of free amino acids
    • G01N33/6812—Assays for specific amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates to a sensor for detecting glutamine using a split glutamine binding protein (QBP) and its use. More specifically, a sensor for detecting glutamine comprising two split domains of a glutamine binding protein, and a sensor using the same. It relates to a method for detecting glutamine and a method for monitoring intracellular glutamine.
  • QBP split glutamine binding protein
  • a specific probe for a target molecule is the most essential part in detecting the target molecule. Since the ligand is capable of interacting with a binding protein (target molecule), a periplasmic binding protein (PBP), lectin, or even a new one can be used to construct the molecular detection module of the sensor system using the binding of the ligand-target molecule. Biosensors using natural ligand-binding proteins such as designed proteins are continuously being researched (Glasgow, AA et al. Science , 366:1024-1028, 2019; Woo, S.-G. et al. Biosens. Bioelectron , 168:112523, 2020).
  • glutamine is one of the molecules with low detection utilization due to limited biosensors (Hunt, JP et al. J. Biotechnol , 325:389-394, 2021). Because glutamine is primarily used as a cellular energy and carbon source in muscle growth and the immune system, optimal glutamine concentration is an important factor in maintaining cellular integrity and function. Immune cells such as lymphocytes, neutrophils, and macrophages require glutamine for proper function, which may provide evidence that changes in glutamine levels are closely related to cancer, diabetes, and neurodegeneration. In particular, in the case of cancer, glutamine consumption rate in cancer cells is much higher than in normal cells, so glutamine metabolism can be an important target for cancer treatment.
  • Amino acids including glutamine
  • glutamine have been proposed as new biomarkers in type 2 diabetes, but accurate and quantitative detection of glutamine in physiological samples has the limitation of using a specific method (high-performance liquid chromatography or amino acid analyzer). These methods require complex pretreatment and sample preparation steps, making accessibility and efficient analysis difficult.
  • Commercially available glutamine colorimetric assay kits are relatively easy to use, but have the disadvantage of low accuracy of results.
  • FRET sensor based on fluorescence-coupled glutamine-binding protein was developed to measure glutamine concentration in vitro and in intracellular environments, but with a novel protein-ligand interaction.
  • the FRET system for FRET requires a rigorous optimization process of various parameters and has the disadvantage of requiring very sensitive and expensive equipment for high spatiotemporal resolution (Dattelbaum, JD & Lakowicz, JR, Anal. Biochem , 291:89-95, 2001; Behjousiar, A., Kontoravdi, C. & Polizzi, K.M. PLoS One , 7:e34512, 2012).
  • a biosensor system in order to manufacture a sensor that can detect glutamine more quickly and conveniently, a biosensor system was developed that recognizes a ligand using a periplasmic binding protein (PBP) and emits fluorescence when bound to the ligand.
  • PBP periplasmic binding protein
  • Q-SHINE Glutamine sensor via Split HINGE-motion binding protein
  • the Q-SHINE system consists of only two protein domains, each containing two split domains of glutamine binding protein, and has the advantage of being able to easily detect glutamine on site without any additional reagents.
  • the purpose of the present invention is to provide a sensor for detecting glutamine comprising two divided domains of glutamine binding protein and a method for detecting glutamine using the same.
  • Another object of the present invention is to provide a sensor for monitoring intracellular glutamine containing two divided domains of glutamine binding protein and a method for monitoring intracellular glutamine using the same.
  • the present invention provides a first domain consisting of a large domain derived from a glutamine binding protein represented by the amino acid sequence of SEQ ID NO: 2 and a C-terminal domain of a reporter protein;
  • a sensor for detecting glutamine comprising a second domain composed of the N-terminal domain of a reporter protein and a small domain derived from a glutamine binding protein represented by the amino acid sequence of SEQ ID NO: 3,
  • the N-terminal domain of the reporter protein is the remaining portion excluding the C-terminal domain of the reporter protein
  • the binding of the large domain derived from the glutamine binding protein and the small domain derived from the glutamine binding protein is induced, which causes the reporter protein C-terminal domain and the reporter protein N-terminal domain to bind and induce a signal. , provides a sensor for detecting glutamine.
  • the glutamine may be L-glutamine.
  • the reporter protein is a fluorescent protein, luciferase, beta-lactamase, SEAP (Secreted embryonic alkaline phosphatase), ubiquitin, and DHFR (Dihydrofolate).
  • TEV protease TEV protease, chorismatemutase, thymidine kinase, APEX2 peroxidase, BioID (Biotin ligase), Cas9 nuclease, Cre recombinase, Cytosine deaminase, Glycinamide ribonucleotide transformylase, HRP (Horse radish peroxidase), T7 RNA polymerase, TET demethyl TET demethylase, Thymidine Kinase, ABL Kinase, Src Kinase, Akt1 Kinase, Lyn Kinase, PTP1B phosphatase , SHP1 phosphatase, or PTPH1 phosphatase.
  • the fluorescent protein is green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), and cyan fluorescent protein.
  • the first domain is represented by the amino acid sequence of SEQ ID NO: 8
  • the second domain is represented by the amino acid sequence of SEQ ID NO: 9
  • the first domain may be represented by the amino acid sequence of SEQ ID NO: 15, and the second domain may be represented by the amino acid sequence of SEQ ID NO: 16.
  • the present invention provides a glutamine detection method comprising reacting a biological sample with the glutamine detection sensor.
  • the present invention provides a first fluorescent protein
  • T2A domain represented by the amino acid sequence of SEQ ID NO: 32;
  • a sensor for intracellular glutamine monitoring comprising a recombinant vector expressing a glutamine detection domain consisting of a small domain derived from a glutamine binding protein represented by the amino acid sequence of SEQ ID NO: 3,
  • the second fluorescent protein N-terminal domain is the remaining portion excluding the second fluorescent protein C-terminal domain
  • a sensor for monitoring intracellular glutamine is provided.
  • the glutamine may be L-glutamine.
  • the first or second fluorescent protein is preferably green fluorescent protein (GFP), yellow fluorescent protein (YFP), or red fluorescent protein.
  • green fluorescent protein, RFP red fluorescent protein
  • CFP cyan fluorescent protein
  • BFP blue fluorescent protein
  • ECFP TagCFP
  • Ds-Red dsRed monomer
  • tetracysteine fluorescent motif SCFP3A
  • sCFP1,2, Cerulean Gmars-T (mMaple)
  • rsEGFP2, sfGFP Folding reporter GFP, EGFP, mKusabira-Green, mKusabira-Green2, sGFP1,2,3, mTSapphire, Photoactivatable GFP, Dronpa, mNeonGreen2, sYFP1 ,2,3, UnaG, EYFP, Venus, sfYFP, Citrine, mKusabira-Orange2, mRFP1, mIris, mEos3.2, CyOFP1,
  • the glutamine detection domain when the first fluorescent protein is green fluorescent protein (GFP) and the second fluorescent protein is mcherry, the glutamine detection domain has the amino acid sequence of SEQ ID NO: 33. It may be displayed or encoded with the base sequence of SEQ ID NO: 35.
  • GFP green fluorescent protein
  • the recombinant vector expressing the glutamine detection domain may be one or more types selected from the group consisting of plasmid vectors, retroviral vectors, and lentivirus-based retroviral vectors.
  • the present invention provides a method for monitoring intracellular glutamine, which includes the step of transforming a cell with a recombinant vector expressing the glutamine detection domain and then measuring a fluorescent signal.
  • the Q-SHINE system of the present invention can potentially be applied as a reagent-free point-of-care diagnosis for a variety of diseases related to glutamine levels.
  • Figure 1 is a schematic diagram showing the Q-SHINE system.
  • Figure 1a is a schematic diagram showing the process of producing a domain derived from glutamine-binding protein.
  • the cleavage site (yellow) was selected at the hinge based on the hinge bending motion of glutamine-binding protein (QBP), and one fragmented domain was selected.
  • the final two QBP individual domains (split QBP_Lg and split QBP_Sm) were prepared by linking the domains with a newly designed linker sequence.
  • Figure 1b is a schematic diagram showing the Q-SHINE system configuration domain and glutamine detection process.
  • the split QBP domain is combined with one of the split mCherry fragments to form split QBP_Lg - mCherry_C (LgC) and mCherry_N - split QBP_Sm (SmN).
  • the Q-SHINE system was constructed.
  • the Q-SHINE system generates fluorescence by binding the cleaved mCherry fragments due to glutamine-induced QBP heterodimerization.
  • Figure 2 is data showing the results of analytical gel filtration chromatography of the Q-SHINE domain.
  • the split QBP_Lg - mCherry_C (LgC) domain is indicated by a solid line
  • the mCherry_N - split QBP_Sm (SmN) domain is indicated by a dotted line.
  • the QBP_Lg domain purified from the Ni column and the eluted fractions from the GPC column (labeled 14 to 24) were loaded on 12% SDS-PAGE and shown in Figure 2b.
  • the molecular weight of the eluted domain was different from the expected value (24.4 kDa and 29.5 kDa for LgC and SmN, respectively), and two separate peaks of LgC (LgC_1 (fraction 15/16)) were used for measurement. and LgC_2(21/22)) were compared.
  • Figure 3 shows data confirming the self-assembly of the Q-SHINE system in the absence of glutamine.
  • the Q-SHINE system of the present invention was compared with Q-SHINE- ⁇ Sm lacking the split QBP_Sm (SmN) portion.
  • Figure 4 is data evaluating the function of the Q-SHINE system.
  • Figure 4a shows data measuring glutamine-dependent BiFC (Bimolecular fluorescence complementation) signals when glutamine was treated at different concentrations and reacted for 2 to 94 hours, and each signal intensity was normalized to wells without glutamine.
  • BiFC Bimolecular fluorescence complementation
  • Figure 4b is data showing the fluorescence intensity of the Q-SHINE system according to wavelength (580 nm to 640 nm, 1 nm increments) when glutamine was treated at different concentrations and reacted for 20 hours.
  • Figure 4c shows data measuring the degree of fluorescence after treating glutamine (Gln), glutamate (Glu), asparagine (Asn), and D-glutamine (D-Gln) at different concentrations to confirm the ligand specificity of Q-SHINE. am.
  • Figure 4d is data showing fluorescence intensity according to glutamine concentration when using the Q-SHINE system applying NanoBiT.
  • Figures 4C and 4D data were normalized to the signal of wells without added amino acids or glutamine. All experiments were repeated three times, and error bars represent standard deviation (SD).
  • Figure 5 is data showing the standard curve of glutamine solution measured with the Q-SHINE system. Although linearity of the signal was observed below 500 ⁇ M glutamine, a non-linear standard fit was used taking into account the fact that physiological glutamine concentrations can exceed 1 mM.
  • Figure 6 shows data comparing a commercial glutamine detection kit and the Q-SHINE system of the present invention when measuring glutamine concentration in mouse serum.
  • Figure 7 shows data measuring the BiFC signal depending on the target substance by applying the Q-SHINE system of the present invention to another ligand (target substance) binding periplasmic binding protein (PBP).
  • CjaA is a cysteine binding protein
  • HBP is a histidine binding protein
  • PEB1 is an aspartate/glutamate binding protein.
  • the mCherry fluorescence intensity by the three proteins was measured in the presence or absence of the corresponding amino acid, and the results were expressed as the mean ⁇ standard deviation (SD) of the fluorescence intensity normalized to the 0mM condition.
  • SD standard deviation
  • FIG. 8 is a schematic diagram showing the structural modeling of genetically encoded Q-SHINE.
  • the Q-SHINE system for intracellular glutamine measurement was prepared by recombining split QBP with GFP and split mCherry fragments and inserting the T2A sequence between GFP-LgC and SmN for equal expression ratios.
  • Figure 9 shows data evaluating the genetically encoded Q-SHINE system in mammalian cells.
  • Figure 9a is data quantifying the mCherry/GFP fluorescence intensity ratio by Q-SHINE-GFP or Q-SHINE-GFP_ ⁇ Sm expressed in HEK293T according to glutamine concentration and reaction time.
  • Figure 9b is a microscopic image of Q-SHINE-GFP or Q-SHINE-GFP_ ⁇ Sm expressed in HEK293T as glutamine concentration increases. An overlay image of the green and red channels is shown, and the scale bar represents 200 ⁇ m.
  • Figure 10 shows data evaluating the genetically encoded Q-SHINE system in plant cells. The mCherry to GFP intensity ratio is indicated.
  • the present invention provides a first domain consisting of a large domain derived from a glutamine binding protein represented by the amino acid sequence of SEQ ID NO: 2 and a C-terminal domain of a reporter protein;
  • a sensor for detecting glutamine comprising a second domain composed of the N-terminal domain of a reporter protein and a small domain derived from a glutamine binding protein represented by the amino acid sequence of SEQ ID NO: 3,
  • the N-terminal domain of the reporter protein is the remaining portion excluding the C-terminal domain of the reporter protein
  • the binding of the large domain derived from the glutamine binding protein and the small domain derived from the glutamine binding protein is induced, which causes the reporter protein C-terminal domain and the reporter protein N-terminal domain to bind and induce a signal.
  • a sensor for detecting glutamine In the presence of glutamine, the binding of the large domain derived from the glutamine binding protein and the small domain derived from the glutamine binding protein is induced, which causes the reporter protein C-terminal domain and the reporter protein N-terminal domain to bind and induce a signal.
  • the glutamine may be L-glutamine.
  • the reporter protein is a fluorescent protein, luciferase, beta-lactamase, SEAP (Secreted embryonic alkaline phosphatase), ubiquitin, DHFR (Dihydrofolate reductase), TEV protease ( TEV protease), chorismatemutase, thymidine kinase, APEX2 peroxidase, BioID (Biotin ligase), Cas9 nuclease, Cre recombinase ), Cytosine deaminase, Glycinamide ribonucleotide transformylase, HRP (Horse radish peroxidase), T7 RNA polymerase, TET demethylase, Thymidine Kinase, ABL Kinase, Src Kinase, Akt1 Kinase, Lyn Kinase, PTP1B phosphatase, SHP1 Phos
  • the fluorescent protein includes green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), and cyan fluorescent protein (cyan fluorescent protein (CFP), blue fluorescent protein (BFP), ECFP, TagCFP, Ds-Red, dsRed monomer, tetracysteine fluorescent motif, SCFP3A, sCFP1,2, Cerulean, Gmars-T (mMaple), rsEGFP2, sfGFP, Folding reporter GFP, EGFP, mKusabira-Green, mKusabira-Green2, sGFP1,2,3, mTSapphire, Photoactivatable GFP, Dronpa, mNeonGreen2, sYFP1,2,3, UnaG, EYFP, Venus, sfYFP , Citrine, mKusabira-Orange2, mRFP1, mIris, mEos3.2, CyOFP1, mScarlet-I, PAmCh
  • the C-terminal fragment of the reporter protein and the N-terminal fragment of the reporter protein are divided from a full sequence reporter protein, and when the reporter protein is mcherry, the first domain is of SEQ ID NO: 8.
  • the second domain may be represented by the amino acid sequence of SEQ ID NO: 9.
  • the first domain may be represented by the amino acid sequence of SEQ ID NO: 15
  • the second domain may be represented by the amino acid sequence of SEQ ID NO: 16.
  • PBP periplasmic binding protein
  • PBP ligand binding protein
  • ligands bind at the interface of the two domains.
  • the conformational energy of PBP (required for the transition from an open to a partially closed state) is strongly influenced by the structural configuration of the hinge region (i.e., mutations in the hinge sequence lead to significant changes in conformational energy) and the ligand binding interface. Mutation of two hinge residues changes the binding affinity for the ligand 50-fold while remaining the same (Seo, MH, et al., Nat. Commun.
  • the sensor component was designed by splitting QBP into two separate domains that, in principle, dimerize only in the presence of glutamine.
  • the split QBP domain the cleaved end of the large domain (Lg) was reconnected by inserting a designed linker (TGNG, SEQ ID NO: 4) to prepare a QBP large domain (SEQ ID NO: 2) that facilitates soluble expression of the domain.
  • TGNG designed linker
  • SEQ ID NO: 2 QBP large domain
  • Sm QBP large domain
  • Each split QBP fragment, Lg and small domain (Sm) was then combined with a split mCherry fragment (C-terminal portion (MC160, SEQ ID NO: 5) and N-terminal portion (MN159, SEQ ID NO: 6)) as reporter elements, respectively.
  • the Q-SHINE system a glutamine detection sensor composed of LGC (first domain, QBP_Lg-mCherry_C, SEQ ID NO: 8) and SmN (second domain, mCherry_N-QBP_Sml, SEQ ID NO: 9), was prepared by recombination.
  • a Gly-Ser linker (GGGSG, SEQ ID NO. 7) was used to connect the split QBP domain and the mCherry fragment.
  • the Q-SHINE domain prepared in the present invention was purified and then analyzed by gel filtration chromatography (GPC). As shown in Figure 2a, both domains (LgC and SmN) were confirmed to move faster than the expected size. . This is presumed to be because the two independent fragments are connected by a flexible linker, and each original segment may become unstable, causing the recombinant protein to become elongated rather than spherical. As shown in Figure 2b, LGC was separated into two individual peaks (LgC_1 and LGC_2), and SmN eluted as a single peak.
  • the self-assembly ability of the Q-SHINE system was evaluated.
  • Bimolecular fluorescence complementation (BiFC) is a technique that divides a fluorescent protein into two fragments. When the two fragments are far apart, they do not emit fluorescence, but when they come close to each other, they combine to form an intact fluorescent protein complex. This is a technique that utilizes the phenomenon of forming fluorescence.
  • BiFC fundamentally relies on the self-assembly tendency of nearby split fluorescent proteins, the self-assembly ability of the Q-SHINE system was compared with the Q-SHINE_ ⁇ Sm (QBP Sm removed) system, which cannot bind glutamine. As a result, as shown in Figure 3, the Q-SHINE system showed slower self-assembly than the Q-SHINE_ ⁇ Sm system in the absence of ligand.
  • the Q-SHINE system in order to confirm whether various reporter proteins can be applied to the Q-SHINE system, the Q-SHINE system was prepared by applying NanoBiT, a luciferase, as a reporter protein to the split QBP domain. .
  • the luminescence signal increased depending on the glutamine concentration.
  • the system using luciferase one of the reporter proteins, has the same sensitivity as mCherry-based Q-SHINE because the sensor components are essentially the same.
  • the mCherry-based Q-SHINE system showed a higher fold change in signal (i.e., higher signal-to-noise ratio) due to the cumulative fluorescence by the irreversibly complemented domains, whereas the NanoBiT-based Q -The SHINE system was confirmed to provide an immediate response in less than 10 minutes.
  • the simple configuration of the Q-SHINE system which includes two protein domains, allows for very simple measurements without pre-processing or expensive equipment, so Q-SHINE can replace other existing glutamine detection systems/sensors.
  • the Q-SHINE system can be applied to detect other amino acids or small molecules.
  • cysteine binding protein CjaA
  • histidine binding protein HBP
  • aspartate/ Glutamate binding protein PEB1
  • methionine binding protein Methionine binding protein
  • DBP glycyl-L-leucine binding protein
  • BtuF vitamin B12 binding protein
  • the present invention relates to a method for detecting glutamine comprising reacting a biological sample with the sensor for detecting glutamine of the present invention.
  • biological sample refers to a sample such as tissue, cells, blood, serum, plasma, saliva, cerebrospinal fluid, or urine, and preferably refers to blood, plasma, or serum.
  • the glutamine detection method using the glutamine detection sensor (Q-SHINE system) of the present invention can be applied not only to the presence of glutamine but also to the measurement of glutamine concentration.
  • the present invention has another consistent aspect,
  • T2A domain represented by the amino acid sequence of SEQ ID NO: 32;
  • a sensor for intracellular glutamine monitoring comprising a recombinant vector expressing a glutamine detection domain consisting of a small domain derived from a glutamine binding protein represented by the amino acid sequence of SEQ ID NO: 3,
  • the second fluorescent protein N-terminal domain is the remaining portion excluding the second fluorescent protein C-terminal domain
  • the present invention relates to a method for monitoring intracellular glutamine, which includes the step of transforming a cell with a recombinant vector expressing the glutamine detection domain and then measuring a fluorescent signal.
  • the first fluorescent protein and the second fluorescent protein are proteins with different fluorescence wavelengths or fluorescence colors, such as green fluorescent protein (GFP), yellow fluorescent protein (YFP), and red fluorescence.
  • Protein red fluorescent protein, RFP), cyan fluorescent protein (CFP), blue fluorescent protein (BFP), ECFP, TagCFP, Ds-Red, dsRed monomer, tetracysteine fluorescent motif ), SCFP3A, sCFP1,2, Cerulean, Gmars-T (mMaple), rsEGFP2, sfGFP, Folding reporter GFP, EGFP, mKusabira-Green, mKusabira-Green2, sGFP1,2,3, mTSapphire, Photoactivatable GFP, Dronpa, mNeonGreen2, sYFP1,2,3, UnaG, EYFP, Venus, sfYFP, Citrine, mKusabira-Orange2, mRFP1, mIr
  • the glutamine detection domain is represented by the amino acid sequence of SEQ ID NO: 33 or the base of SEQ ID NO: 35. Can be coded as a sequence.
  • the recombinant vector expressing the glutamine detection domain may be one or more types selected from the group consisting of plasmid vectors, retroviral vectors, and lentivirus-based retroviral vectors.
  • a genetically encoded Q-SHINE system was prepared for monitoring intracellular glutamine.
  • GFP and the split mCherry C-terminal fragment (GFP-QBP_Lg-mCherry_C) were connected to the QBP large domain, and the GFP-QBP_Lg-mCherry_C domain and the mCherry N-QBP_Sm domain were used for equal expression.
  • a recombinant vector (Q-SHINE recombinant vector) expressing the Q-SHINE system with self-cleaving T2A peptide inserted was prepared.
  • the Q-SHINE-GFP system of the present invention identifies the mCherry signal and It was confirmed that glutamine could be detected.
  • Example 1 Production of a sensor for glutamine detection
  • the Q-SHINE domain was designed to be divided based on hinge bending motion analysis (FIG. 1).
  • QBP glutamine-binding protein
  • QBP_Lg one domain
  • Linker modeling was performed using RosettaRemodel as the default option (Huang, PS et al. PLoS One, 6:e24109, 2011), and loop closure attempts were performed 10,000 times for various loop lengths from 2 to 5. It has been done. Then, the designed loop was filtered using RosettaScript's LoopAnalyzerMover (Fleishman, SJ et al. PLoS One, 6:e20161, 2011). The Rosetta total score, which summarizes loop quality, was used to select a final loop sequence that stabilized the fusion domain, while also prioritizing shorter loop lengths to minimize flexibility. Through the linker modeling, a linker having the TGNG (SEQ ID NO: 4) sequence was selected and inserted between the divided parts of QBP_Lg.
  • TGNG SEQ ID NO: 4
  • QBP_Lg large domain derived from a glutamine-binding protein represented by the amino acid sequence of SEQ ID NO: 2
  • QBP_Sm small domain derived from a glutamine-binding protein represented by the amino acid sequence of SEQ ID NO: 3
  • each split QBP fragment, QBP_Lg domain and QBP_Sm domain was coupled with the reporter protein split mCherry fragment, mCherry C-terminal domain (SEQ ID NO: 5, MC160) and mCherry N-terminal domain (SEQ ID NO: 6, MN159), respectively. It was recombined.
  • the split QBP domain and mCherry fragment were connected using a Gly-Ser linker (GGGSG, SEQ ID NO: 7).
  • Q-SHINE domain sequence information domain order sequence number Q-SHINE first domain QBP_Lg-mCherry_C) MHHHHHHLVVATDTAFVPFEFKQGDLYVGFDVDLWAAIAKELKLDYELKPMDFSGIIPALQTKNVDLALAGITITDERKKAIDFSDGYY TGNG QQYGIAFPKGSDELRDKVNGALKTLRENGTYNEIKKWFGTEA GGGSG GALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEG RHSTGGMDELYK SEQ ID NO: 8 second domain (mCherry_N-QBP_Sm) MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVLRGTNF
  • a Q-SHINE domain consisting of LGC (first domain, Split QBP_Lg - mCherry_C) represented by the amino acid sequence of SEQ ID NO: 8 and SmN (second domain, mCherry_N - Split QBP_Sm) represented by the amino acid sequence of SEQ ID NO: 9 was designed (Table 1).
  • Q-SHINE_ ⁇ Sm QBP Sm removal
  • nucleotide sequences complementary to the pET21a (Novagen) plasmid are placed at both ends of the LGC domain gene encoded by the base sequence of SEQ ID NO: 10 and the SmN domain gene encoded by the base sequence of SEQ ID NO: 11. It was synthesized by IDT gBlocks® or TWIST BIOSCIENCE to include this.
  • the pET21a plasmid was linearized by digestion with NdeI and XhoI enzymes, and then the LGC domain gene and SmN domain gene were cloned into the linear pET21a vector using the In-Fusion HD cloning kit (Takara Bio).
  • the pET21a plasmid in which the LGC domain and the SmN domain were cloned, was transformed into E. coli BL21(DE3) cells, and then protein expression was induced using 0.5mM IPTG (isopropyl b-D-1-thiogalactopyranoside) at 18°C.
  • IPTG isopropyl b-D-1-thiogalactopyranoside
  • the protein was purified from the soluble cell lysate using His60 Ni Superflow resin (Takara Bio), and the concentration was measured using NanoDrop.
  • the purified protein on the Ni column was loaded onto a Superdex 75 column equilibrated with 20mM Tris-HCl (pH 8.0, 150mM NaCl).
  • Molecular mass standards include coalbumin (75 kDa), ovalbumin (44 kDa), carbonic anhydrase (29 kDa), and ribonuclease (13.7 kDa).
  • Q-SHINE_ ⁇ Sm the LGC domain gene encoded by the nucleotide sequence of SEQ ID NO: 10 and the mCherry N-terminal domain gene encoded by the nucleotide sequence of SEQ ID NO: 12 were inserted into the pET21a plasmid in the same manner as above, and then inserted into E. coli BL21 ( It was prepared by transformation into DE3) cells.
  • both domains (LgC and SmN) were confirmed to move faster than the expected size. This is presumed to be because the two independent fragments are connected by a flexible linker, and each original segment may become unstable, causing the recombinant protein to become elongated rather than spherical.
  • the LGC domain was separated into two individual peaks (LgC_1 and LGC_2), and the SmN domain eluted as a single peak.
  • the self-assembly ability of the Q-SHINE system manufactured in Example 1 was evaluated. Since the bimolecular fluorescence complementation (BiFC) technique fundamentally relies on the self-assembly propensity of nearby split fluorescent proteins, the self-assembly ability of the Q-SHINE system cannot be combined with glutamine, Q-SHINE_ ⁇ Sm (QBP Sm removed). compared to the system.
  • BiFC bimolecular fluorescence complementation
  • Q-SHINE was added to a 6-well plate (Nunc) so that the concentration of the LGC domain and SmN domain was 5 ⁇ M, and Q-SHINE_ ⁇ Sm was added to the mCherry N-terminal fragment from which the LGC domain and the QBP_Sm portion was removed at a concentration of 5 ⁇ M. This was added to a 6-well plate (Nunc). Then, glutamine was added and the reaction was allowed to take place in the dark at room temperature for 1 to 5 hours. The emission spectrum of mCherry was read from 600 nm to 620 nm in 1 nm increments using an Infinite M1000 (TECAN) at 580 nm excitation.
  • TECAN Infinite M1000
  • the Q-SHINE system showed slower self-assembly than the Q-SHINE_ ⁇ Sm system in the absence of ligand.
  • the Q-SHINE system could easily detect 1 ⁇ M glutamine in the mixture, and the emission peak was observed at 610 nm, which is the reported value for native mCherry protein.
  • the Q-SHINE system showed very specific detection ability only for glutamine and could not detect other amino acids except glutamine.
  • the Q-SHINE system of the present invention exhibits high detection sensitivity even in the concentration range of hundreds of micromolar to millimolar, which corresponds to physiological glutamine levels.
  • the Q-SHINE_NanoBiT system was prepared by applying NanoBiT as a reporter protein to the split QBP domain.
  • the Q-SHINE_NanoBiT system is a BiT small domain (SmBiT) represented by the amino acid sequence of SEQ ID NO: 13 and a BiT large domain (LgBiT) represented by the amino acid sequence of SEQ ID NO: 13 and the LGC domain and SmN domain of Example 1-1, respectively. ) was designed to be connected. That is, the Q-SHINE_NanoBiT system consists of a first domain (QBP_Lg - SmBiT) represented by the amino acid sequence of SEQ ID NO: 15 and a second domain (LgBiT - QBP_Sm) represented by the amino acid sequence of SEQ ID NO: 16.
  • the first domain (QBP_Lg - SmBiT) encoded by the nucleotide sequence of SEQ ID NO: 17 and the second domain (LgBiT - QBP_Sm) encoded by the nucleotide sequence of SEQ ID NO: 18 were added to the pET21a plasmid. After insertion, it was prepared and purified by transformation into E. coli BL21(DE3) cells.
  • Q-SHINE_NanoBiT domain sequence information domain order sequence number first domain (QBP_Lg-SmBiT) MHHHHHHLVVATDTAFVPFEFKQGDLYVGFDVDLWAAIAKELKLDYELKPMDFSGIIPALQTKNVDLALAGITITDERKKAIDFSDGYYTGNGQQYGIAFPKGSDELRDKVNGALKTLRENGTYNEIKKWFGTEAGSSGGGGSGGGGSSGMVTGYRLFEEIL SEQ ID NO: 15 second domain (LgBiT-QBP_Sm) MVFTLEDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIQRIVRSGENALKIDIHVIIPYEGLSADQMAQIEEVFKVVYPVDDHHFKVILPYGTLVIDGVTPNMLNYFGRPYEGIAVFDGKKIVTGTLWNGNKIIDERLITPDGSMLFRVTINSGSSGGGGSGGGGSSGGGLLVMVKANNNDVKSVKLD
  • the final concentration of the two domains of Q-SHINE_NanoBiT was prepared to be 5 ⁇ M each, and then mixed with glutamine at a concentration of 0 to 5mM for 10 minutes at room temperature in the dark.
  • 25 ⁇ l of Nano-Glow Live Cell Reagent included in Nano-Glo Live Cell Assay (Promega) was added to each well, mixed gently for 10 seconds, and analyzed at a single time point using the GloMax Navigator System (Promega). Luminescence was measured immediately for each time point. As a result, as shown in Figure 4D, it was confirmed that the luminescence signal increased depending on the glutamine concentration. In other words, it was confirmed that glutamine could be effectively detected even when luciferase was applied as a reporter protein to the Q-SHINE system of the present invention.
  • mice Female C57BL/6 mice aged between 6 and 12 weeks were purchased from Orient-Bio (Korea). Mice were raised in accordance with the institutional guidelines for laboratory animals, and the experimental plan was approved by the Animal Care Committee of the Korea Institute of Science and Technology (KIST) (approval number KIST-2021-09-104). To collect serum samples, the mouse was anesthetized and an abdominal incision was made to collect blood samples, then centrifuged at 300g for 10 minutes at 4°C, and at least 200 ⁇ l of serum was collected and stored at -80°C.
  • KIST Animal Care Committee of the Korea Institute of Science and Technology
  • Q-SHINE showed a dramatic signal contrast at glutamine concentrations in the range of 100 to 1000 ⁇ M and that physiological glutamine concentrations generally range from 200 to 1400 ⁇ M
  • 20 ⁇ l serum was used to make up 100 ⁇ l of total reaction mixture.
  • a final glutamine concentration of several hundred ⁇ M was used.
  • the fluorescence intensity from the wells was read by excitation at 587 nm and emission at 610 nm after 5 h of reaction, and the glutamine concentration in mouse serum was determined based on a standard curve obtained by plotting the values of the standard glutamine solution.
  • the standard curve was reacted by mixing the two domains of Q-SHINE in the same manner as above by varying the concentration of the glutamine solution dissolved in distilled water for the standard curve.
  • the standard curve was fitted using Origin 2020's nonlinear curve fitting method (Hill1 function), and the glutamine concentration of the serum sample was calculated using 'Find X from Y' based on the fitted graph.
  • the glutamine concentration in mouse serum was simultaneously determined by the EnzyChrom Glutamine Assay Kit (EGLN-100, BioAssay Systems, Hayward, USA) according to the manufacturer's protocol.
  • Example 1 To further test whether the system of the present invention can be applied in the same way to other ligand binding PBPs, the method of Example 1 was used to bind cysteine binding protein (CjaA), histidine binding protein (HBP), and aspartate/glutamate binding protein. (PEB1), methionine binding protein (MetQ), glycyl-L-leucine binding protein (DBP), or vitamin B12 binding protein (BtuF) were each prepared by fusing them with the split mCherry domain. Each sequence information is shown in Table 4 below.
  • CjaA_Lg-mCherry_C MHHHHHHCGGNSDSKTLNSLDKIKQNGVVRIGVFGDKPPFGYVDEKGNNQGYDIALAKRIAKELFGDENKVQFVLVEAANRVEFLKSNKVDIILANFTQTPQRAEQVDFCSGSGPAVKKGDKELKEFDNLIIKLGQEQFFHKAYDETLKAHFGDDVKADDVVIEGGKIGSSGGGGSGGGALKGEIKQRLLKDGGH YDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELY SEQ ID NO: 19 second domain (mCherry_N-CjaA_Sm) MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLS
  • GFP SEQ ID NO: 31
  • GFP-QBP_Lg-mCherry_C split mCherry C-terminal fragment
  • GFP-QBP_Lg-mCherry_C split mCherry C-terminal fragment
  • the Q-SHINE-GFP_ ⁇ Sm domain (GFP-QBP_Lg-mCherry_C-T2A-mCherry N, SEQ ID NO: 34), in which the Sm domain was not expressed, was designed and manufactured.
  • Q-SHINE-GFP system sequence information domain order sequence number Q-SHINE-GFP (GFP-QBP_Lg-mCherry_C-T2A-mCherry N-QBP_Sm)
  • a Q-SHINE-GFP domain expression recombinant vector was prepared by inserting the Q-SHINE-GFP domain gene encoded by the nucleotide sequence of SEQ ID NO: 35 into the vector, and as a control, Q-SHINE-GFP_ ⁇ Sm encoded by the nucleotide sequence of SEQ ID NO: 36
  • a Q-SHINE-GFP_ ⁇ Sm domain expression recombinant vector was prepared by inserting the domain gene into the vector.
  • the pcDNA3.1(+) vector was linearized using PCR amplification using a pair of primers (pcDNA_Amp_FW and pcDNA_Amp_RV).
  • pcDNA_Amp_FW a pair of primers
  • mCherry SEQ ID NO: 38
  • the whole expression cassette of the pcDNA plasmid amplified with primers (QBP_Split_FW_pGO and QBP_Split_RV or QBP_Split_RV_Mut) was cloned into the pTALCOMT plasmid for Agrobacterium-mediated transformation. did.
  • Example 8 Monitoring glutamine in mammalian cells using the Q-SHINE-GFP system
  • the Q-SHINE-GFP domain expression recombinant vector and the Q-SHINE-GFP_ ⁇ Sm domain expression recombinant vector prepared in Example 7 were respectively used in HEK293T cells. It was transfected and cultured.
  • HEK293T Human embryonic kidney 293T cells were purchased and used from ATCC (American Type Culture Collection). Cell lines were tested for mycoplasma contamination and used within 30 passages only. HEK293T was cultured in MDEM (Gibco) medium supplemented with 10% (v/v) FBS (heat-inactivated FBS, Gibco), 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin at 5% CO 2 and 37°C. .
  • the mammalian expression vector was transiently transfected using Lipofectamine3000 (Invitrogen). Specifically, for intracellular BiFC experiments, HEK293T cells were inoculated into 6-well TC-plates (Thermo) and then incubated in DMEM (10% FBS and 1% PS) to reach a cell concentration of 4 to 50% confluency. Added) medium was used and cultured overnight at 37°C and 5% CO 2 conditions.
  • the medium was changed to glutamine-free DMEM (added 10% FBS and 1% PS), and then HEK293T cells were transfected with a plasmid containing the Q-SHINE domain using Lipofectamine3000 (Invitrogen). . 16 hours after transfection, the medium was replaced with a final medium containing 0 to 2.0mM glutamine.
  • the present invention the possibility of monitoring glutamine in plant cells using the Q-SHINE-GFP system was confirmed. Since the endogenous glutamine concentration in plant cells is in the range of 2.5 to 20mM, which is excessive for measurement, the present invention simply uses Q-SHINE-GFP as Q Compared to -SHINE-GFP_ ⁇ Sm, it was confirmed whether Q-SHINE can operate in the plant system when expressed.
  • Agrobacterium-mediated transient expression analysis was performed using methods known in the art (Lee, JH, et al., Front. Plant Sci. , 11 :1617, 2020). Specifically, Agrobacterium tumefaciens LBA4404 cells were adjusted to OD 600 0.4 in resuspension solution (10mM MgCl 2 , 10mM MES-KOH (pH 5.6), 100 ⁇ M acetosyringone) and grown in 4- to 5-week-old tobacco cells. (Nicotiana tabacum cv. Samsun) leaves were infiltrated.
  • Plants were grown and cultured at 24/21°C (day/night), 16-h photoperiod, 500 ⁇ mol/m 2 /s light intensity, and 80% relative humidity. Five days after infiltration, the fluorescence intensity of leaves was measured using an LSM5 Zeiss confocal laser scanning microscope with ZEN image analysis software (Carl Zeiss, Jena, Germany). Excitation/emission spectra are 488nm/493-598nm for GFP and 594nm/599-650nm for mCherry.
  • the Q-SHINE-GFP system of the present invention can detect glutamine in plant cells by identifying the mCherry signal.
  • the Q-SHINE system of the present invention can potentially be applied as a reagent-free point-of-care diagnosis for a variety of diseases related to glutamine levels.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Biotechnology (AREA)
  • Pathology (AREA)
  • Microbiology (AREA)
  • General Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Biophysics (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un capteur pour détecter la glutamine à l'aide d'une protéine de liaison à la glutamine (QBP) scindée et son utilisation et, plus précisément : un capteur pour détecter la glutamine, comprenant deux domaines scindés d'une protéine de liaison à la glutamine; un procédé pour détecter la glutamine à l'aide de celui-ci; et un procédé pour surveiller la glutamine dans une cellule. En conséquence de la mesure de la concentration de glutamine dans une solution ou un sérum murin à l'aide d'un système Q-SHINE, qui est un capteur pour détecter la glutamine à l'aide d'une protéine de liaison à la glutamine de la présente invention, il a été confirmé que le capteur présente une sensibilité et une spécificité supérieure, comparables à des kits de dosage de glutamine commerciaux. Ainsi, le système Q-SHINE de la présente invention peut potentiellement être appliqué à des analyses de biologie délocalisée sans réactif pour diverses affections associées aux niveaux de glutamine. Il a également été confirmé que la concentration intracellulaire de glutamine peut être surveillée en temps réel à la fois dans des cellules de mammifère et de plante à l'aide du système Q-SHINE génétiquement codé de la présente invention.
PCT/KR2023/001429 2022-05-31 2023-01-31 Capteur pour détecter la glutamine à l'aide d'une protéine de liaison à la glutamine scindée et son utilisation WO2023234513A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2022-0066824 2022-05-31
KR1020220066824A KR20230167247A (ko) 2022-05-31 2022-05-31 분할된 글루타민 결합 단백질을 이용한 글루타민 검출용 센서 및 이의 용도

Publications (1)

Publication Number Publication Date
WO2023234513A1 true WO2023234513A1 (fr) 2023-12-07

Family

ID=89025137

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2023/001429 WO2023234513A1 (fr) 2022-05-31 2023-01-31 Capteur pour détecter la glutamine à l'aide d'une protéine de liaison à la glutamine scindée et son utilisation

Country Status (2)

Country Link
KR (1) KR20230167247A (fr)
WO (1) WO2023234513A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20180070985A (ko) * 2016-12-19 2018-06-27 서강대학교산학협력단 L-글루타민 검출용 fret 센서 및 이를 이용하는 l-글루타민 검출 방법

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20180070985A (ko) * 2016-12-19 2018-06-27 서강대학교산학협력단 L-글루타민 검출용 fret 센서 및 이를 이용하는 l-글루타민 검출 방법

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATTELBAUM, J.D. ; LAKOWICZ, J.R.: "Optical Determination of Glutamine Using a Genetically Engineered Protein", ANALYTICAL BIOCHEMISTRY, ACADEMIC PRESS, AMSTERDAM, NL, vol. 291, no. 1, 1 April 2001 (2001-04-01), Amsterdam, NL , pages 89 - 95, XP027472793, ISSN: 0003-2697, DOI: 10.1006/abio.2001.4998 *
DONALDSON TERAYA; IOZZINO LUISA; DEACON LINDSAY J.; BILLONES HILBERT; AUSILI ALESSIO; D'AURIA SABATO; DATTELBAUM JONATHAN D.: "Engineering a switch-based biosensor for arginine using aThermotoga maritimaperiplasmic binding protein", ANALYTICAL BIOCHEMISTRY, ACADEMIC PRESS, AMSTERDAM, NL, vol. 525, 1 March 2017 (2017-03-01), Amsterdam, NL , pages 60 - 66, XP029959439, ISSN: 0003-2697, DOI: 10.1016/j.ab.2017.02.021 *
SEO MOON-HYEONG, LIM YUN, KIM JI YUL, JUNG YOUN HEE, LEE JAE HOON, BAEK MIN SEOK, JUNG JE HYEONG, KIM HO-YOUN, LEE WOOKBIN, PARK K: "Q-SHINE: a versatile sensor for glutamine measurement via ligand-induced dimerization", BIORXIV, 14 February 2023 (2023-02-14), XP093116777, DOI: 10.1101/2022.06.28.497868 *
WADA AKIRA, MIE MASAYASU, AIZAWA MASUO, LAHOUD PEDRO, CASS ANTHONY E. G., KOBATAKE EIRY: "Design and Construction of Glutamine Binding Proteins with a Self-Adhering Capability to Unmodified Hydrophobic Surfaces as Reagentless Fluorescence Sensing Devices", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, AMERICAN CHEMICAL SOCIETY, vol. 125, no. 52, 1 December 2003 (2003-12-01), pages 16228 - 16234, XP093116771, ISSN: 0002-7863, DOI: 10.1021/ja036459l *

Also Published As

Publication number Publication date
KR20230167247A (ko) 2023-12-08

Similar Documents

Publication Publication Date Title
Cabantous et al. A new protein-protein interaction sensor based on tripartite split-GFP association
US7015310B2 (en) Oxidation reduction sensitive green fluorescent protein variants
Lam et al. Directed evolution of APEX2 for electron microscopy and proximity labeling
Shyu et al. Identification of new fluorescent protein fragments for bimolecular fluorescence complementation analysis under physiological conditions
EP0815257B1 (fr) Procede de detection de substances biologiquement actives
Feng et al. Bright split red fluorescent proteins for the visualization of endogenous proteins and synapses
Tammsalu et al. Proteome-wide identification of SUMO modification sites by mass spectrometry
WO2012128580A1 (fr) Polypeptides hydrosolubles constitués par des modules à répétition, leur procédé de préparation et procédé de préparation d'un polypeptide spécifique de cible et d'analyse de son activité biologique
WO2013048185A2 (fr) Procédé de formation d'un nano-agrégat réversible de protéines à l'aide de lumière dans une cellule
JP6682532B2 (ja) 神経毒ポリペプチドの生物学的活性を測定する方法
WO2012115454A2 (fr) Procédé pour concentrer des cellules qui sont génétiquement modifiées par des nucléases
WO2019177345A1 (fr) Procédé de détection ultrasensible de biomarqueurs multiples
WO2023234513A1 (fr) Capteur pour détecter la glutamine à l'aide d'une protéine de liaison à la glutamine scindée et son utilisation
Kaczmarczyk et al. A novel biosensor reveals dynamic changes of C-di-GMP in differentiating cells with ultra-high temporal resolution
Dacres et al. Direct comparison of bioluminescence-based resonance energy transfer methods for monitoring of proteolytic cleavage
Yasuno et al. Effects of N‐Glycosylation Deletions on Cypridina Luciferase Activity
US9017962B2 (en) Intracellular reporters
Kim et al. Opthis identifies the molecular basis of the direct interaction between csl and smrt corepressor
WO2014129808A1 (fr) Procédé de criblage et de quantification et de l'activité d'une enzyme de biosynthèse d'isoprène
CN102174640B (zh) 量化和/或分析键特异性多泛素链的方法
US20070022484A1 (en) Probes for analyzing interaction between proteins and method of analyzing interaction between proteins using the same
WO2017135568A1 (fr) Mimétique d'anticorps pouvant être activé de manière réversible et son utilisation
WO2021256582A1 (fr) Biocapteur pour la détection de tryptophane, comprenant un facteur d'activation de transcription et un commutateur toehold
WO2017204561A2 (fr) Polynucléotide recombinant codant pour un polypeptide comprenant une fraction rapporteur, une fraction de substrat et une fraction de déstabilisation, cellule hôte le comprenant et son utilisation
WO2019022467A1 (fr) Procédé de quantification d'acides aminés au moyen d'un système de synthèse de protéines sans cellules

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23816186

Country of ref document: EP

Kind code of ref document: A1