WO2018048063A1 - Method for quantitatively measuring interaction between membrane proteins in living cells - Google Patents

Method for quantitatively measuring interaction between membrane proteins in living cells Download PDF

Info

Publication number
WO2018048063A1
WO2018048063A1 PCT/KR2017/005544 KR2017005544W WO2018048063A1 WO 2018048063 A1 WO2018048063 A1 WO 2018048063A1 KR 2017005544 W KR2017005544 W KR 2017005544W WO 2018048063 A1 WO2018048063 A1 WO 2018048063A1
Authority
WO
WIPO (PCT)
Prior art keywords
membrane protein
protein
target
egfr
proteins
Prior art date
Application number
PCT/KR2017/005544
Other languages
French (fr)
Korean (ko)
Inventor
류성호
이남기
김도현
박소연
김동균
권용훈
정민규
Original Assignee
포항공과대학교 산학협력단
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 포항공과대학교 산학협력단 filed Critical 포항공과대학교 산학협력단
Publication of WO2018048063A1 publication Critical patent/WO2018048063A1/en

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
    • 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
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • 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
    • 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
    • G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention relates to a method for quantitatively measuring the interaction between membrane proteins in living cells.
  • interactions in cells can be analyzed using fluorescence resonance energy transfer measurement, fluorescence recovery after photobleaching, and protein-fragment complementarity methods.
  • fluorescence resonance energy transfer measurement fluorescence resonance energy transfer measurement
  • fluorescence recovery after photobleaching fluorescence recovery after photobleaching
  • protein-fragment complementarity methods have a problem in that they cannot provide quantitative information for determining equilibrium constants such as dissociation constants, which are absolute values indicating the tendency of protein complexes to dissociate into a single protein.
  • Patent Document 1 The present inventors have presented a method for analyzing the membrane protein binding aspect of living cells through the Republic of Korea Patent Application No. 10-2014-0059027 (Patent Document 1).
  • Patent Document 2 the Republic of Korea Patent Application No. 10-2015-0060619 (Patent Document 2) has suggested a mechanical analysis method of the membrane protein diffusion through the real-time analysis technology of super resolution single particle tracking data.
  • Single particle tracking is a technique that provides information about the diffusion of individual proteins in the cell membrane of living cells.
  • the single particle tracking method has the potential advantage of uncovering dynamic and fast molecular-level functions based on the inherent proliferation of proteins, many factors, such as protein interactions, microdomain entrapment, and cytoskeleton settling, Because they appear mixed in pathway data, there is a limited approach to identifying specific biological phenomena in the interpretation of these data.
  • the above problem can be solved by introducing two different color quantum dots in a single particle tracking method to observe the overlap of these two paths, but two membrane proteins labeled with different colors have the same time. This approach is disadvantageous in analyzing the number of interacting molecules because the probability of meeting at the same location is extremely low.
  • Patent Document 1 Korean Patent Application No. 10-2014-0059027
  • Patent Document 2 Korean Patent Application No. 10-2015-0060619
  • the present invention seeks to provide a method for the quantitative analysis of transient interactions between membrane proteins in living single cells.
  • a method for analyzing the interaction between a target membrane protein and a binding partner membrane protein for the target membrane protein in living cells
  • a capture probe having binding capacity to both the substrate and the target membrane protein is treated with the cells of analysis to immobilize the target membrane protein on the substrate,
  • cell to be analyzed means a cell used for analyzing the interaction between a specific membrane protein and a membrane protein that can interact with it.
  • the cells to be analyzed are living cells, and any kind thereof may be used.
  • the cell under analysis can be a cell that selectively overexpresses the target membrane protein. Overexpression of the target membrane protein is not essential for the assay method of the present invention.
  • the present invention aims to quantitatively analyze the transient interaction between a target membrane protein and a binding partner membrane protein for the target membrane protein on living cells.
  • the "target membrane protein” is a membrane protein located on the cell membrane, and means a membrane protein which wants to grasp the interaction with other membrane proteins, and the kind thereof is not particularly limited.
  • target membrane protein is also expressed as “bait protein”.
  • target membrane protein andbait protein are used interchangeably.
  • EGFR is used as the target membrane protein, and the change in the movement of the membrane protein interacts with it.
  • membrane proteins other than EGFR can also be analyzed as the target membrane protein.
  • binding partner membrane protein means a protein that interacts with a target membrane protein.
  • Membrane proteins known to or expected to interact with the target membrane protein can be set as binding partner membrane proteins and analyzed for interaction with the target membrane protein.
  • the "binding partner membrane protein” is also expressed as “prey protein” (Prey).
  • binding partner membrane protein and “feed protein (Prey)” are used interchangeably.
  • the "target membrane protein” and the "binding partner membrane protein” for the target membrane protein include an integral membrane protein, a peripheral membrane protein, a transmembrane protein, It may be selected from the group consisting of membrane glycoprotein (membrane glycoprotein) and lipid anchored membrane protein (lipid anchored membrane protein).
  • the "target membrane protein” and the "binding partner membrane protein” for the target membrane protein may be labeled by a "detectable moiety" to track the movement of the membrane protein in the cell membrane.
  • the “detectable moiety” makes it possible to grasp the presence, the position, the amount of the "target membrane protein” and the “binding partner membrane protein” with respect to the target membrane protein.
  • Detectable moieties for tracking membrane proteins are well known in the art and may be, for example, fluorescent, luminescent, chemiluminescent, radioactive moieties.
  • the membrane protein may be one labeled by the fluorescent moiety as a detectable moiety.
  • membrane proteins may be provided to facilitate expression by expression in the form of fusion proteins with fluorescent proteins.
  • the membrane protein may be a fusion protein with a tag to which an organic fluorescent dye may be bound, and a fluorescent material is labeled on a tag of the fusion protein.
  • tags to which the organic fluorescent dyes may be combined include SNAP tags and CLIP tags.
  • a fluorescent moiety is labeled on a membrane protein by expressing a SNAP tag or a CLIP tag on a membrane protein and binding a fluorescent substance to a terminal of a benzylguanidine derivative or O2-benzylcytosine derivative that binds to these tags.
  • labeling by a probe that specifically binds to a membrane protein may be implemented by a fluorescent moiety in a form in which a fluorescent material is bound thereto.
  • EGFR labeled with EGFR-mEos3.2 having the mEos3.2 protein bound to the C terminus of EGRF, or bound with the SNAP tag to the N terminus of EGFR is labeled with a target membrane of EGFR.
  • Examples used for labeling proteins and / or binding partner membrane proteins are shown.
  • the membrane protein may be labeled via binding to an antibody labeled by a detectable moiety.
  • the fluorescent protein is fused with a membrane protein is Green Fluorescent Protein (GFP) series, Blue fluorescent protein (BFP) series, Cyan series, Yellow Fluorescent Protein (YFP) series, Red Fluorescent Protein (RFP) series, Orange It may be one or more selected from the group consisting of series, Far-red series, Near-IR, photoactivatable protein, photoconvertible protein, and photoswitchable protein.
  • GFP Green Fluorescent Protein
  • BFP Blue fluorescent protein
  • YFP Yellow Fluorescent Protein
  • RFP Red Fluorescent Protein
  • Orange It may be one or more selected from the group consisting of series, Far-red series, Near-IR, photoactivatable protein, photoconvertible protein, and photoswitchable protein.
  • the fluorescent protein is enhanced green fluorescent protein (EGFP), Emerald, Superfolder GFP, Azami green mWasabi, TagGFP, AcGFP, T-sapphire, mUKG, Clover, mNeonGreen, enhanced blue fluorescent protein (EBFP), EBFP2, Azurite , mTagBFP, mKalama1, Sirius, enhanced cyan fluorescent protein (ECFP), monomeric ECFP (mECFP), Cerulean, mTurquoise, mTurquoise2, CyPet, TagCFP, mTFP1 (Teal), SCFP3A, monomeric Midoriishi Cyan, EYFP (enhanced yellow fluorescent protein) Topaz, Benus, mCitrine, YPet, TagYFP, PhiYFP, mBanana, SYFP2, mRuby, mRuby2, mApple, mStrawberry, mRFP1, mCherry, mRa
  • Fusion protein of membrane protein and fluorescent protein refers to a covalent complex formed by genetic fusion or chemical bonding of membrane protein and fluorescent protein.
  • genetic fusion means a linear, covalent linkage formed through the genetic expression of a DNA sequence encoding a protein.
  • the preparation of the fusion protein can be carried out by known methods, for example, the fusion protein can be prepared using genetic recombination techniques.
  • Cells expressing the fusion protein can be prepared by transforming the expression vector.
  • the expression vector may be a plasmid vector, a virus or a cosmid vector.
  • Host cells expressing the fusion protein are either calcium phosphate method or calcium chloride / chloride described in Sambrook, J., et al., Molecular Cloning, A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory, 1. 74, 1989. Rubidium, electroporation, electroinjection, chemical treatment such as PEG, gene gun, and transfection such as virus transfection Can be prepared.
  • a cell expressing a fusion protein by transforming an expression vector including a fusion protein of a target membrane protein and a fluorescent protein into a host cell using lipofectamine, fugene, or metafectin. You can prepare.
  • the fluorescent material may be an organic fluorescent dye, the kind is not particularly limited.
  • organic fluorescent dyes are CF660R, Alexa Flour 647, Atto 488, Alexa Flour 488, Dy505, Rhodamine 123, Atto 520, Dy 530, ATTO 532, Alexa Fluor 532, Fluorescein, FITC, Cy2, Cy3B, Alexa Flour 568 , TAMRA, Cy3, Cy3.5, SNAP-Cell TMR-Star, Atto 565, Atto 590, Alexa Fluor 647, Cy5, Atto 647, Atto 647N, Dyomics 654, Atto 655, TMP-ATTO 655, Atto 680, Cy5.
  • substrate means a plate, a well, or the like used for immobilizing the membrane protein of the cell to be analyzed and observing the immobilization and diffusion of the membrane protein.
  • the substrate may be a slide glass or cover glass.
  • the substrate according to the present invention is not limited to the material, but it is preferable to easily detect a detectable moiety such as a fluorescent substance labeled to track the movement of the membrane protein.
  • capture probe means a probe used to immobilize a target membrane protein on a substrate.
  • the capture probe can be an antibody, antigen-binding fragment, affibody, or aptamer.
  • the capture probe can be provided at a height such that proper contact between the membrane protein and the substrate can occur.
  • a secondary antibody may be additionally used to provide an appropriate height to the antibody used as the capture probe.
  • Capture probes according to the invention have the ability to bind both the substrate and the target membrane protein. This is to obtain the diffusion coefficient distribution of binding partner membrane proteins before and after the treatment of the capture probes.
  • capture probes can be applied to both the substrate and the target membrane protein. It is easy to design to have a binding capacity for.
  • the substrate may be a neutravidin, streptavidin, or avidin is bound. Methods of binding neutravidin, streptavidin, or avidin on a substrate via covalent bonds are well known in the art.
  • diffusion coefficient refers to the average velocity of molecules to diffuse, and in the present invention is an index indicating the mobility of membrane proteins in the cell membrane.
  • the diffusion coefficient of the membrane protein can be obtained by detecting the trajectory in the cell membrane of the membrane protein through single particle tracking (SPT).
  • SPT single particle tracking
  • Single particle tracking is a technique of observing the movement of a single particle in a medium and expressing the coordinates of the movement over time as a trajectory, and analyzing the movement pattern and heterogeneity (Saxton, MJ, Jacobson, K). Single-particle tracking: Applications to membrane dynamics.Annual Review of Biophysics and Biomolecular Structure 1997; 26 (373-399).
  • TIRF microscope is a total reflection microscope, which is suitable for studying the cell membrane and its surroundings because it is possible to observe the fluorescence in a specific region below 200 nm by excluding the interference by other light by using the total reflection phenomenon of light.
  • the TIRF microscope is used to obtain an image of a membrane protein at a time-dependent position and to express it in orbit to obtain a diffusion coefficient.
  • the diffusion coefficient of the membrane protein can be calculated using the following Equation 1 and Equation 2.
  • the diffusion coefficient of a single binding partner membrane protein single particle can be obtained through the single particle tracking analysis to obtain the locus of the binding partner membrane protein single particle, and can be obtained through Equation 1 and Equation 2.
  • the trajectory of a membrane protein single particle is obtained by obtaining a plurality of membrane protein images showing the change of membrane protein position over time through single particle tracking analysis, detecting the position of the membrane protein from the image, and concatenating the detected positions. .
  • the position of the membrane protein over time i.e., coordinates
  • the movement of the coordinates is one target membrane protein It can be expressed as the trajectory of.
  • a diffusion coefficient for one trajectory may be obtained.
  • the time for detecting fluorescence (the time taken to image the membrane protein particles at one coordinate) to determine the position of one membrane protein particle can be changed according to the analysis conditions, for example, about 30 To 70 ms, for example 50 ms.
  • the coordinates required when one target membrane protein particle makes one trajectory is not limited thereto, but preferably five or more.
  • the number of trajectories of the membrane protein single particles used in the single particle trace analysis may be from 3000 to 10,000, preferably 5,000 or more. The number of trajectories for obtaining convergent diffusion coefficient values while reducing uncertainty may vary depending on cell type or condition, so the number of trajectories for appropriate analysis can be adjusted according to the situation.
  • the calculation of the diffusion coefficient uses a trajectory over a certain length, and the fit of the linear function measures the diffusion coefficient with the step size between the coordinates of the target membrane protein particles more than two frames (since the fit of the linear function is possible with two points). To increase the accuracy. These diffusion coefficients can be averaged over the entire trajectory to calculate the average diffusion coefficient of the membrane protein in each cell.
  • the diffusion coefficient of the binding partner membrane protein may be obtained by Fluorescence recovery after photobleaching (FRAP) or Fluorescence Correlation Spectroscopy (FCS).
  • FRAP Fluorescence recovery after photobleaching
  • FCS Fluorescence Correlation Spectroscopy
  • FCS can measure the diffusion coefficient of fluorescence in a liquid by measuring fluctuations in fluorescence intensity over time and analyzing auto-correlation. The intensity of fluorescence optically observed in a very small focal space varies randomly with time around the mean value, and this random fluctuation shows information that the phosphor enters and exits the focal space as Brownian motion occurs. It contains.
  • Diffusion coefficient distribution means a histogram of diffusion coefficients at the level of a single molecule.
  • the horizontal axis represents the diffusion coefficient and the vertical axis represents the number of single molecules having the corresponding diffusion coefficient or the ratio of the single molecules to the whole after standardization.
  • the diffusion coefficient of the binding partner membrane protein will be lower when viewed at the single molecule level.
  • the cell membrane is a mixture of a plurality of target proteins and a plurality of binding partner membrane proteins that exist in a heterogeneous (heterogenous) state.
  • the target membrane protein and the binding partner membrane protein both exist in the form of a population of single molecules in heterogeneous states and can be classified into subpopulations according to the diffusion coefficient of each single molecule.
  • the diffusion coefficient distribution of the target membrane protein undergoes a change when compared to the pretreatment of the capture probe, which is directly immobilized on the substrate by the capture probe.
  • the capture probe which is directly immobilized on the substrate by the capture probe.
  • binding partner membrane proteins that interact with a subset of target membrane proteins immobilized on a substrate are part of a population of binding partner membrane proteins that have been freely roaming on the cell membrane, and some of these binding partner membrane proteins It is called a subpopulation.
  • the diffusion coefficient of the binding partner membrane protein interacting with it is reduced at the single molecule level, which is the diffusion coefficient
  • the number of high subpopulations is reduced and the number of subpopulations with low diffusion coefficients is expressed. This change in diffusion coefficient distribution is more pronounced as the binding partner membrane protein tends to interact with the target membrane protein.
  • an increase in the number of subpopulations having a low diffusion coefficient in the diffusion coefficient distribution of the binding partner membrane protein after treatment of the capture probe means that a temporary interaction with the target membrane protein, that is, complex formation, has occurred.
  • the interaction between the target membrane protein and the binding partner membrane protein is "temporary", and the binding partner protein temporarily forms a complex with the target membrane protein and then dissociates.
  • the method of analyzing the interaction of the membrane protein according to the present invention makes it possible to find the dissociation constant of the binding partner protein for the target membrane protein.
  • the method for analyzing the interaction of a membrane protein measures dissociation constant between a target membrane protein and a binding partner membrane protein for the target membrane protein from a change in diffusion coefficient distribution following treatment of the capture probe. It may further include.
  • the "dissociation constant” refers to the tendency of the target membrane protein and the binding partner membrane protein to form a complex and then dissociate into a single protein.
  • the dissociation constant between the target membrane protein and the binding partner membrane protein for the target membrane protein can be obtained by the following Equation 3, Equation 4 or Equation 5.
  • [I], [R], and [RI] are the concentrations of the target membrane protein, the concentration of the binding partner membrane protein for the target membrane protein, and between the target membrane protein and the binding partner membrane protein for the target membrane protein, respectively. The concentration of the complex is shown.
  • K D [R] 0 (1- ⁇ R ) ([I] 0- [RI]) / [R] 0 ⁇ R
  • [R] 0 , [I] 0 , and ⁇ R are the concentrations of the initial target membrane protein and binding partner membrane protein before the interaction occurs, respectively, after binding probe membrane treatment, compared to before the capture probe treatment. The percentage of subpopulations of proteins.
  • ⁇ R is the subpopulation ratio of binding partner membrane protein increased after treatment compared to before capture probe treatment, and [I 0 ] represents the absolute concentration of immobilized target membrane protein.
  • the dissociation constant is defined as the ratio between the concentration of the target membrane protein, the binding partner membrane protein and the concentration of the complex between the target membrane protein and the binding partner membrane protein for the target membrane protein as shown in [Equation 3].
  • ⁇ R which represents the subpopulation ratio of the binding partner membrane protein increased after treatment as compared to before the capture probe treatment
  • ⁇ R which represents the subpopulation ratio of the binding partner membrane protein increased after treatment as compared to before the capture probe treatment
  • ⁇ R can be obtained by directly comparing the ratio of subpopulations in the diffusion coefficient distribution before and after the capture probe. More specifically, it is possible to calculate the ratio of subpopulations to the entire population by drawing a standardized distribution of the diffusion coefficients measured at the single molecule level and calculating the subarea for each subpopulation. Compared with before the capture probe, the numerical value of ⁇ R can be obtained by calculating the degree to which the proportion of the subpopulations having the low diffusion coefficient increases in the distribution coefficient distribution after the treatment.
  • the total single-molecules with the diffusion coefficient distribution before and after the capture probe treatment must be the same. Standardize The diffusion coefficient distribution is divided into peaks with relatively high diffusion coefficients and peaks with low diffusion coefficients that can be considered to have little diffusion motion and are fitted to a Gaussian graph for each peak. By calculating the ratio of each graph of the total area 1 can be calculated. The increase in the amount of membrane protein interacted before and after the capture probe treatment can be confirmed by using a ratio of peaks having a low diffusion coefficient among them.
  • the area of the peak having the lowest diffusion coefficient in the Gaussian graph was 0.2, respectively, and changed to 0.5 after the capture probe was processed.
  • the concentration of the complex between the target membrane protein, the binding partner membrane protein, the target membrane protein, and the binding partner membrane protein for the target membrane protein in [Equation 3] can be reexpressed as shown in [Equation 4] using ⁇ R. If the concentration of the target membrane protein is much higher than the concentration of the complex between the target membrane protein and the binding partner protein, it can be defined simply as shown in [Equation 5].
  • the method for analyzing the interaction of membrane proteins according to the present invention can be utilized for screening candidate drugs that can effectively act on the target membrane proteins by confirming changes in membrane protein interactions following treatment of candidate drugs.
  • Membrane proteins are a major target for drug development, and more than 50% of all currently developed drugs target membrane proteins (Overington, JP, How many drug targets are there ?, Nature reviews.Drug discovery. 5, 993 -996, 2006).
  • isoprene a drug for the treatment of heart blocks or bradycardia
  • insulin a diabetes drug
  • is known as a drug that targets the insulin receptor Peter Imming. Et al. Drugs, their targets and the nature and number of drug target.Nature reviews.Drug discovery, 2006).
  • the ErbB family plays an important role in cancer, and the importance of developing anticancer agents that target it is already known (Eric K. Rowinsky. THE ERBB FAMILY: Targets for Therapeutic Development against Cancer and Therapeutic Strategies Using Monoclonal Antibodies) and Tyrosine Kinase Inhibitors.Annu. Rev. Med. 55, 433-57, 2004), for example, cetuximab, a colorectal cancer drug, is a drug targeting the ErbB family. Therefore, researches on binding to membrane proteins and ligands, etc. can be applied to the development of drugs that specifically act on the target membrane protein, specifically used as a method for screening drugs for the target membrane protein.
  • a method for screening a candidate drug for a target membrane protein comprising analyzing a change in the interaction between the target membrane protein and a binding partner membrane protein for the target membrane protein in living cells following treatment of the candidate drug,
  • the diffusion coefficient distribution of the binding partner membrane protein before and after the capture probe is obtained, and the diffusion coefficient distribution of the binding partner membrane protein is changed by the diffusion coefficient of the binding partner membrane protein before and after the capture probe treatment in the candidate drug-free group. Selecting candidate drugs that affect the interaction with the target membrane protein in comparison to the change in distribution.
  • the invention also relates to the invention.
  • a method for screening a candidate drug for a target membrane protein comprising analyzing a change in the interaction between the target membrane protein and a binding partner membrane protein for the target membrane protein in living cells following treatment of the candidate drug,
  • the change in the dissociation constant of the binding partner membrane protein following the candidate drug treatment was compared with the change in the binding partner membrane protein dissociation constant before and after the capture probe treatment in the candidate drug-free group to influence the interaction with the target membrane protein. Affecting the selection of candidate drugs
  • Candidate drugs affecting the target membrane protein may be used one or more from the group consisting of compounds, nucleic acids, sugars, carbohydrates, lipids, peptides and proteins.
  • the protein may be, for example, an antibody, wherein the antibody is a concept comprising all or part of all, such as a half-immunoglobulin fragment, which selectively cuts only a Fab portion or a hinge portion.
  • the candidate may be a nucleic acid, wherein the nucleic acid is a concept comprising both oligonucleotides or c-DNAs comprising 2-200 bases.
  • the candidate may be treated in the cells in an appropriate amount to analyze the interaction between the target membrane protein and the binding partner membrane protein, and the treatment may be performed according to methods known in the art.
  • a cell culture solution containing 0.01 to 1000 ug / ml of the candidate material can be directly treated to the cells of analysis placed on the substrate.
  • the candidate drug is the interaction between the target membrane protein and the binding partner membrane protein. It can be considered as the main factor affecting the action. If the change in the distribution coefficient distribution or the dissociation constant of the binding partner membrane protein by the treatment of the candidate drug is at the significance level, for example, such a change is at least 2%, at least 3%, at least 5%, In the case of 10% or more and 20% or more, the candidate may be determined as a drug that affects the interaction between the target membrane protein and the binding partner membrane protein.
  • an antibody specific for a target membrane protein as a capture probe was used to immobilize only a specific target membrane protein on a substrate and allow other membrane proteins to move unaffected.
  • the membrane protein that interacts with the target membrane protein that is, the binding partner membrane protein, temporarily stops freely moving membrane proteins while interacting with the immobilized target membrane protein.
  • the diffusion coefficient Based on the diffusion coefficient, only the path data by the temporary interaction can be distinguished and interpreted.
  • Co-II Co-ImmunoImmobilization
  • the target membrane protein in a method for analyzing an interaction between a target membrane protein and a binding partner membrane protein for the target membrane protein in living cells, is treated by treating a target probe protein with a capture probe for the target membrane protein.
  • Can be calculated on the substrate and the dissociation constant for the interaction can be calculated by measuring the change in the distribution coefficient distribution of the binding partner membrane protein for the target membrane protein and thus the target membrane protein and the binding partner membrane protein for the target membrane protein. It is possible to measure the interaction between them quantitatively.
  • FIG. 1 shows a schematic diagram of a method for quantitatively examining the transient interaction between membrane proteins in living single cells based on the diffusion coefficient.
  • the proportion of this subpopulation is calculated as the subpopulation ratio (pR) with a low diffusion coefficient increased in the distribution coefficient distribution of the feed protein.
  • the dissociation constant (K D ) of the complex RI formed through the transient interaction between the binding partner membrane protein R and the target membrane protein I measures the ratio ⁇ R of the increased immobilized subpopulation after immobilizing protein I on the coverslip. Can be calculated.
  • COS7 cells expressing mEos3.2-EGFR were divided into low (# 1), middle (# 2), and high (# 3) according to the expression level of mEos3.2 fluorescence intensity.
  • Figure 4 shows the results confirming the efficiency of the degree of immobilization when using various antibodies.
  • the ratio of immobilized EGFR in each COS7 cell, HEK293 cell, HeLa cell, CHO-K1 cell expressing EGFR-mEos3.2 before and after treatment with the EGFR specific antibody was measured. It can be seen that the ratio of immobilized EGFR in all cell lines is similar.
  • Figure 6 shows the results of experiments with molecular specific immobilization in the cell membrane of living cells.
  • FIG. 7 shows the results of measuring the dissociation constants of transient interactions in EGFR pre- homodimer formation .
  • the dissociation constant of EGFR pre-homogenous dimers in COS7 cells is independent of the expression level of SNAP-EGFR and its mean value is about 2727 ⁇ 126 molecules / ⁇ m 2 .
  • (h) Graph showing the ratio of binding / non-binding ratio of SNAP-EGFR to the ratio of immobilized subpopulations of EGFR-mEos3.2 and the ratio of immobilized subpopulations of EGFR-mEos3.2 according to the amount of expression of SNAP-EGFR. to be.
  • the red line represents the pathway of one of the EGFR-mEos3.2 monomolecules
  • the white dot represents SNAP-EGFR immobilized by SNAP tag specific antibodies. It can be seen that the position of the immobilized SNAP-EGFR and part of the pathway of the temporarily immobilized EGFR-mEos3.2 coexist at exactly the same position.
  • the fluorescence signal was quantified from the original image using the ImageJ program, and the protein concentration was calculated by dividing the total fluorescence signal by the fluorescence signal of a single molecule.
  • the protein concentration obtained from CF660R-SNAP and Cetuximab Alexa flour 647 for EGFR of the same cell was 0.91 ⁇ 0.13.
  • the ratio of immobilized subpopulations after treatment of SNAP tag specific antibodies to SNAP-EGFR expressed in COS7 cells shows a positive correlation with expression levels.
  • One dot in the data represents information from one cell.
  • MEos3.2-EGFR expressed in COS7 cells was immobilized on the coverslip using mEos3.2 protein specific antibodies.
  • the degree of phosphorylation of EGFR with or without EGF was measured using a phosphorylated antibody of Y1068 amino acid of EGFR and a secondary antibody combined with Alexa 647. Fluorescence signals were quantified from the original image using the ImageJ program.
  • the immobilization of EGFR the phosphorylation of EGFR was significantly increased in the EGF-treated environment compared to NT.
  • EGFR Homodimeric The effect of direct binding of tyrosine phosphorylation inhibitor on dissociation constant was confirmed.
  • TKI tyrosine phosphorylation inhibitor
  • co-immuno immobilization uses 'bait' proteins (target membrane proteins) immobilized by specific antibodies coated on coverslips.
  • the 'feed' proteins interaction candidate proteins, ie binding partner membrane proteins
  • FOG. 1A interaction candidate proteins, ie binding partner membrane proteins
  • Equation 3 If the amount of immobilized bait protein is significantly greater than the amount of complex, it can be assumed that the absolute concentration of the bait protein is equal to the initial concentration when the dynamic equilibrium is reached. Therefore, in [Equation 3], [R] / [RI] is substituted by the ratio of the feed protein that is not bound to the bait protein, which is a dimensionless value, and does not need the absolute concentration information of the feed protein.
  • the second order bimolecular response equation can be calculated as a pseudo first order reaction. This value is determined by determining the ratio ⁇ R of the feed protein with the diffusion coefficient immobilized by the interaction between the two increased after immobilization, as compared with before the fixation of the bait protein. It can substitute and use in ratio. Therefore, Equation 3 for obtaining the dissociation constant may be converted as shown in Equation 5 in the experimental design proposed by the present invention. (More substitution procedures can be found in the methods.)
  • EGFR epithelial growth factor receptor
  • a coverslip coated with an EGFR specific antibody 3-mercaptopropyltrimethoxysilane was used to prepare a coverslip in which a thiol reactor was activated.
  • neutravidin with maleimide is activated to induce covalent bonds between the reactors to make neutravidin-bound coverslips, and finally to EGFR-specific biotin-bound Antibodies were treated.
  • EGFR (EGFR-mEos3.2) linked to the C-terminus at the C-terminus in COS7 cells, and then immobilized EGFR resulting from treatment of EGFR-specific antibodies with biotin bound using a multi-particle tracking method
  • the proportion of subpopulations was measured (FIG. 2A).
  • the ratio of immobilized EGFR was not as high as ⁇ 61% despite treatment with a sufficient amount of antibody capable of immobilizing not only the intentionally expressed EGFR but also the endogenously expressed EGFR (FIG. 2D).
  • the distance between the cell membrane and the cover slip surface reaching 50 nm or more is about 30 nm from the cover slip surface, and the height of the EGFR is estimated to be about 15 nm from the cover slip surface. It was judged that EGFR was not sufficiently immobilized because it could only physically bind to only some surfaces in the cells adhered to the coverslip. Therefore, we wanted to increase the height from the coverslip by treating one additional antibody (secondary antibody) between neutravidin and EGFR specific antibodies (FIG. 2B). The expected height using the secondary antibody was about ⁇ 45 nm, resulting in immobilization of> 97.3% of EGFR in COS7 cells that adhered well to the coverslip (FIG. 2D).
  • secondary antibody neutravidin and EGFR specific antibodies
  • the EGFR (SNAP-EGFR) with the SNAP tag coupled to the N-terminus of EGFR and the EGFR (mEos3.2-EGFR) with the mEos3.2 protein bound to the SNAP tag-specific and mEos3.2 protein-specific antibodies, respectively.
  • Immobilization efficiency for the present invention was examined.
  • SNAP-EGFR and mEos3.2-EGFR showed almost complete immobilization efficiency against SNAP tag-specific and mEos3.2 protein-specific antibodies, respectively, but EGFR-mEos3.2 with mEos3.2 linked to the C terminus of EGFR .2 unaffected by protein specific antibodies (FIGS. 4D-F).
  • the beta2 adrenergic receptor ( ⁇ 2-AR), which is known to not physically interact with EGFR, was selected (Fig. 6a) to select EGFR labeled with the CF660R fluorescent dye and ⁇ 2-AR labeled with the mEos3.2 fluorescent protein based on the SNAP tag.
  • ⁇ 2-AR beta2 adrenergic receptor
  • the position error ⁇ can be derived from the average diffusion coefficient ⁇ ⁇ 2 / t, which is used to estimate the position error of the CF660R dye and the mEos3.2 protein.
  • the diffusion coefficient criterion that distinguishes is defined.
  • the diffusion coefficient of the ⁇ 2-AR subpopulations decreased slightly by ⁇ 13.4%, this is presumably due to the increased congestion between the cell membrane and the coverslip, and this decrease was used to quantify the proportion of immobilized subpopulations. There was a slight effect.
  • the immobilization efficiency increased by ⁇ 8.7% on average, which is interpreted as a significant level of ErbB2 interacts with EGFR.
  • the expression was performed by overexpressing these membrane proteins relative to the expression level of the intrinsic EGFR. Since only ErbB2 and ErbB3 are known to interact with EGFR, it can be inferred that the heterodimers formed by ErbB2 by interacting with EGFR are particularly strong compared to ErbB3.
  • SNAP tag-specific antibodies were treated to cells expressing SNAP-EGFR and EGFR-mEos3.2 to specifically target SNAP-EGFR only. Immobilized (FIG. 7A).
  • Other protein tags or fluorescent proteins may also be used instead of SNAP tags, but SNAP tags were used to obtain high-density pathway data and to confirm complete SNAP-EGFR immobilization.
  • the diffusion coefficient distribution of SNAP-EGFR shows that the moving pathways are fully immobilized ( ⁇ 95.2%) after antibody treatment, whereas EGFR-mEos3.2 is approximately 22.7% partially immobilized. Only conversion was shown (FIG. 7C-E).
  • the increased proportion of immobilized subpopulations of EGFR-mEos3.2 can be seen here to eventually indicate the amount of EGFR pre-homogenous dimers.
  • the temporary immobilization of EGFR-mEos3.2 forms a dimer with SNAP-EGFR. May be (FIG. 8).
  • the concentration of SNAP-EGFR expressed on the surface of the COS7 cell was determined by dividing the total fluorescence signal on the surface by a single molecule signal (FIG. 7F).
  • a total reflection fluorescence image of SNAP-EGFR labeled with CF660R fluorescent dye was obtained, where the intensity of the total fluorescence signal is the total amount of SNAP-EGFR at the surface of the COS7 cell.
  • the photobleaching turned off the fluorescence signal until the SNAP protein could be observed at the individual molecular level, and the total reflection fluorescence image was taken again.
  • one step of photobleaching was used to select fluorescence signals of a single molecule, and their average was taken to determine a single molecule fluorescence signal generated from one CF660R fluorescent dye.
  • the total number of SNAP-EGFR was corrected in consideration of the ratio of SNAP-EGFR that was immobilized but not detected because CF660R fluorescence was not activated (FIG. 9).
  • the calculated SNAP-EGFR concentration was 650 ⁇ 54 molecules / ⁇ m 2 . Because membrane proteins only diffuse from side to side on two-dimensional cell membranes, the definition of molar concentration in the cell membranes is not clear.
  • COS7 cells expressing both SNAP-EGFR and EGFR-mEos3.2 were starved for 4 hours, and then dissociation constants for dimerization of EGFR with or without EGF were measured using co-immuno immobilization.
  • the expression level of SNAP-EGFR was maintained at least 10 times higher than the expression level of EGFR-mEos3.2 to meet the conditions of the similar primary response.
  • the dissociation constant of EGFR pre-dimer without EGF was determined to be ⁇ 7244 molecules / ⁇ m 2
  • the dissociation constant of EGFR dimer formed by EGF was measured to be ⁇ 369 molecules / ⁇ m 2 .
  • the treatment of EGF may have contributed to a significant increase in the phosphorylation of EGFR by EGF by about 20 times the dissociation constant.
  • the dissociation constant of the EGFR predimer in the cell-hungry environment was found to be about 2.6 times higher than the dissociation constant under normal conditions (10% FBS) where the cells grow (Fig. 7g), which is indirect. It means that phosphorus elements can affect the formation of dimers of EGFR.
  • EGFR In the absence of EGF in the area of cholesterol-rich cell membranes, EGFR tends to exist in a structure that is folded and bound to extracellular sites, thus eliminating cholesterol facilitates the action of unfolding the folded structure of EGFR and consequently favors dimer formation. It is assumed to create an environment. However, at the same time, cholesterol in the cell membrane seems to function to stabilize the EGFR dimerization induced by EGF, so Nystatin treatment may be considered to partially increase the dissociation constant of EGFR dimer by EGF. This dual nature of cholesterol to EGFR dimerization widens the difference in the dimerization of EGFR with and without ligand, which is an important function of the lipid raft for ligand-induced EGFR signaling in cell membranes. It can be said that.
  • the optimal expression level of the bait protein can be determined analytically through Equation 5 depending on the dissociation constant of the interaction.
  • the efficiency of immobilizing the bait protein is an important factor in terms of measurement accuracy, but it has already been confirmed that even in highly overexpressed (less than one million receptor) cells, this immobilization efficiency does not drop significantly (FIG. 3). It is expected to be possible to measure. Therefore, even the interactions that form transiently 10 times or more than the EGFR pre-dimer can be analyzed by co-immuno immobilization.
  • This joint immunofixation technique is generally applicable to the interaction of various cell membrane proteins with a wide range of binding strengths in living cells. Identifying the quantitative interaction characteristics of membrane proteins in various physiological environments will provide a new perspective for an in-depth understanding of the dynamic nature of membrane protein interactions in living cells.
  • PcDNA3.1 / SNAP tag-EGFR was obtained by obtaining the SNAP tag portion from the pSNAPf vector (N9183S, New England Biolabs) to create a fusion protein to which the SNAP tag was linked, and inserting it into the pcDNA3.1 / mEos3.2-EGFR vector. .
  • All plasmids used are the following references 1 (Kim, DH et al. Analysis of Interactions between the Epidermal Growth Factor Receptor and Soluble Ligands on the Basis of Single-Molecule Diffusivity in the Membrane of Living Cells. Angew. Chem. Int. Ed Engl. 54 , 7028-32 (2015).
  • the sources of antibodies and reagents are as follows: mAb 199.12 and Alexa fluor 647 linked anti-mouse antibodies are available from Invitrogen; mAb 528 and mAb R-1 are purchased from Santa Cruz; Rabbit anti-Mouse IgG and biotin-linked EGFR antibodies are available from Thermo Scientific; anti-mEos3.2 antibodies are available from Badrilla; anti-phosphorylated EGFR antibody (Y1068) was purchased from abcam; the anti-actin antibodies are available from MP Biomedicals; Erlotinib and lapatinib are purchased from Roche; Nystatin and latrunculin B were purchased from Sigma Aldrich.
  • COS7, HEK293, and HeLa cells were purchased from the American Type Culture Collection (ATCC), Dulbecco's modified Eagle medium (DMEM, Lonza) medium containing 10% FBS (Gibco) at 37 ° C, 5% CO2 and 95% humidity. Cells were cultured. In the same way, CHO-K1 cells purchased from ATCC were cultured at 37 ° C, 5% CO2, and 95% humidity in a medium containing 1: 1 mixed DMEM with F-12 medium (Thermo Scientific) and 10% FBS. It was. All cells were transformed using Lipofectamine LTX (Invitrogen) according to the manufacturer's instructions.
  • ATCC American Type Culture Collection
  • DMEM Dulbecco's modified Eagle medium
  • FBS Gibco
  • FBS FBS
  • All cells were transformed using Lipofectamine LTX (Invitrogen) according to the manufacturer's instructions.
  • the coverslips were first washed with chloroform / methanol (50:50) for 24 hours and then stored in ethanol. After drying, the coverslip was oxidized in a plasma chamber (Femto Science) for 1 minute and 30 seconds, and in methanol, a silane containing 4.5% of deionized water, 0.9% of acetic acid and 2.5% of 3 mercapto-pro-pyulrimethoxy silane (S10475, Fluorochem). Into the citation solution and reacted at 4 °C for about 9-12 hours.
  • the treated coverslips were washed three times with a solution of Phosphate Buffered Saline, and then reacted with a activated maleimide reactor (31007, Thermo Scientific) at room temperature for 1 hour in a maleimide reactor diluted in fibronectin solution at a concentration of 50 ⁇ g / ml.
  • a maleimide reactor diluted in fibronectin solution at a concentration of 50 ⁇ g / ml.
  • the biotin conjugated goat anti-rabbit IgG H & L antibody (ab7089, abcam) or biotin conjugated goat anti-mouse IgG Fc antibody (A16088, Invitrogen) was further reacted for 1 hour prior to settlement.
  • DMEM Thermo Scientific
  • CF660R (92134, biotium) with succinimidyl ester reactor was reacted with BG-NH2 (New England Biolabs) in dimethylformamide (DMF) for about 9-12 hours at 30 ° C according to the manufacturer's instructions. After reaction the solvent was evaporated in vacuo and the product was dissolved in distilled water after purification by HPLC.
  • BG-CF660R or SNAP-Surface Alexa Fluor 647 (New England Biolabs), which were previously prepared for fluorescent labeling in EGFR, and then seated on an antibody-coated coverslip.
  • Alexa Fluor 647 a reductant is added to the media together with a deoxidant as shown in Ref.
  • Fluorescence imaging was performed on an objective-based TIRF fluorescence microscope (IX-71, Olympus) equipped with an XYZ axis automated stage.
  • the laser and fluorescence filter used in the experiment were used as in Reference 1.
  • All fluorescence images were obtained using an electron amplification coupled soju device (EM-CCD / iXon3 897, Andor Technology).
  • a tube lens of 1.43x was used with a 1.6x amplifier mounted on the microscope to obtain higher magnification images.
  • Live cell imaging equipment (Chamlide TC-A, Live cell instrument) was used to maintain live cells during imaging. All instrument manipulation and imaging data was obtained using MetaMorph (Molecular Devices) and a hand-written MATLAB (The MathWorks) plug-in.
  • Diffusion coefficients were calculated using the mean squared displacements (MSD) of Equations 1 and 2, assuming that the particles had a normal diffusion phenomenon in two-dimensional space.
  • the dissociation constant is defined as in Equation 3 below.
  • [R], [I], and [RI] represent the concentrations of the individual proteins R, I, and complex RI, respectively.
  • the diffusion coefficient of membrane protein R is measured as a probability density function, and the immobilization ratio of R increases as the complexes of R and I are formed.
  • the immobilization ratio is expressed by ⁇ R
  • the dissociation constant may be expressed as Equation 4 below.
  • [R 0 ] and [I 0 ] are the initial concentrations of proteins R and I.
  • Equation 5 the equation of the dissociation constant can be simplified as shown in Equation 5 below.
  • the dissociation constant can be calculated using two values, [I 0 ], which is the initial concentration of I, and ⁇ R , which is an increased immobilization ratio after antibody treatment specific to I.
  • the mEos3.2 fluorescent protein expressed with the cell membrane protein was irradiated with a laser at 488 nm wavelength, and the organic fluorescent dyes CF660R and Alexa Flour 647 labeled with the membrane protein were irradiated with a laser at 642 nm wavelength, respectively.
  • the total expression level was quantified by measuring the average fluorescence signal using at least five positions in the same cell and removing background signals from three or more images using the ImageJ program. After the imaging for path tracking was completed, the fluorescent molecules were subjected to photobleaching with a high power laser to observe at a single molecule level.
  • the total concentration of a particular membrane protein expressed in a single cell was calculated by dividing the total fluorescence intensity by the degree of expression by the fluorescence intensity of a single molecule.

Abstract

The present invention relates to a method for analyzing an interaction between a target membrane protein and a binding partner membrane protein for the target membrane protein in living cells. According to the present invention, in the method for analyzing an interaction between a target membrane protein and a binding partner membrane protein for the target membrane protein in living cells, the dissociation constant for the interaction can be calculated by treating cells to be analyzed with a capture probe for the target membrane protein to immobilize the target membrane protein on a substrate and measuring a change of the diffusion coefficient distribution of the binding partner membrane protein for the target membrane protein. Therefore, the interaction between the target membrane protein and the binding partner membrane protein for the target membrane protein can be quantitatively measured.

Description

살아있는 세포에서 막 단백질 간의 상호작용을 정량적으로 측정하는 방법How to quantitatively measure the interaction between membrane proteins in living cells
본 발명은 살아있는 세포에서 막 단백질 간의 상호작용을 정량적으로 측정하는 방법에 관한 것이다. The present invention relates to a method for quantitatively measuring the interaction between membrane proteins in living cells.
생체 분자들 사이의 일시적이고 약한 상호작용은 살아있는 세포가 활발하게 유지되기 위해 필수적인 기능을 수행한다. 대표적으로 키나아제, 포스파타아제, 프로테아제를 포함하는 효소 반응은 특정한 기질과의 일시적인 상호작용을 통해 이루어지며, 세포 내 골격이나 소포체를 포함하는 세포 구조체 또한 일시적인 상호작용을 통해 역동적으로 생성되고 해체된다. 그러나 기존에는 살아있는 세포 내에서 매우 빠르게 발생하는 일시적인 상호작용을 정량적으로 측정하는 데 있어서 기술적 한계가 존재하기 때문에 생체 분자들 사이의 상호작용에 대해 여전히 많은 부분이 알려지지 않고 있다. 시험관 내에서 일시적인 상호작용을 하는 두 단백질을 생화학적 교차연결시킴으로써 영구적으로 결합하도록 한 후 공동 면역 침강법을 이용하게 되면 두 단백질의 결합 여부에 대해 측정이 가능하지만 정량적인 정보는 잃게 된다. 또한, 형광 공명 에너지 전달 측정, 광 퇴색 후 형광 회복 측정, 단백질-단편 상보성 방법을 이용하여 세포 내에서 상호작용을 분석할 수 있다. 그러나 이러한 방법들은 단백질 복합체가 단일 단백질로 해리되는 경향성을 가리키는 절대적인 수치인 해리상수와 같은 평형 상수를 결정하는 정량적인 정보를 제공할 수 없다는 문제점을 가지고 있다.Temporary and weak interactions between biomolecules perform essential functions to keep living cells active. Representative enzymatic reactions, including kinases, phosphatase, and proteases, typically occur via transient interactions with specific substrates, and cellular constructs, including intracellular frameworks and endoplasmic reticulum, are also dynamically generated and disassembled through transient interactions. However, there is still a great deal of unknown about the interactions between biomolecules because of technical limitations in quantitatively measuring transient interactions that occur very quickly in living cells. If two proteins with temporary interactions in vitro are permanently bound by biochemical crosslinking, then co-immunoprecipitation can be used to determine whether the two proteins bind, but quantitative information is lost. In addition, interactions in cells can be analyzed using fluorescence resonance energy transfer measurement, fluorescence recovery after photobleaching, and protein-fragment complementarity methods. However, these methods have a problem in that they cannot provide quantitative information for determining equilibrium constants such as dissociation constants, which are absolute values indicating the tendency of protein complexes to dissociate into a single protein.
본 발명자들은 대한민국 특허출원 제10-2014-0059027호(특허문헌 1)를 통해 살아있는 세포내 막 단백질 결합 양상을 분석하는 방법을 제시한 바 있다. 또한, 대한민국 특허출원 제10-2015-0060619호(특허문헌 2)를 통해 초해상도 단일 입자 추적 데이터의 실시간 분석 기술을 통한 막 단백질 확산의 역학적 분석 방법을 제시한 바 있다.The present inventors have presented a method for analyzing the membrane protein binding aspect of living cells through the Republic of Korea Patent Application No. 10-2014-0059027 (Patent Document 1). In addition, the Republic of Korea Patent Application No. 10-2015-0060619 (Patent Document 2) has suggested a mechanical analysis method of the membrane protein diffusion through the real-time analysis technology of super resolution single particle tracking data.
단일 입자 추적 방법은 살아있는 세포의 세포막에서 개별 단백질들의 확산에 대한 정보를 제공하는 기법이다. 비록 단백질이 가진 고유의 확산성을 기반으로 역동적이고 빠른 분자 수준의 기능들을 밝히는 데 있어서 단일 입자 추적 방법이 가지고 있는 잠재적 강점이 있지만, 단백질 간의 상호작용, 미세영역 얽매임, 세포골격 정착 등 다양한 요소들이 경로 데이터에 혼합되어 나타나기 때문에 이러한 데이터의 해석에 있어서 특정한 생물학적 현상을 구분하여 접근하는 것이 제한적이다. 단일 입자 추적 방법에 두 가지의 서로 다른 색을 지닌 양자점(quantum dot)을 도입하여 이 두 가지 경로가 겹쳐짐을 관찰함으로써 위에서 발생한 문제를 해결할 수 있으나 각기 다른 색으로 표지된 두 개의 막 단백질이 동일한 시간에 동일한 위치에서 만날 확률은 극단적으로 낮기 때문에 이러한 접근법은 상호작용하는 분자들의 수를 분석하는 데 있어서 불리하다.Single particle tracking is a technique that provides information about the diffusion of individual proteins in the cell membrane of living cells. Although the single particle tracking method has the potential advantage of uncovering dynamic and fast molecular-level functions based on the inherent proliferation of proteins, many factors, such as protein interactions, microdomain entrapment, and cytoskeleton settling, Because they appear mixed in pathway data, there is a limited approach to identifying specific biological phenomena in the interpretation of these data. The above problem can be solved by introducing two different color quantum dots in a single particle tracking method to observe the overlap of these two paths, but two membrane proteins labeled with different colors have the same time. This approach is disadvantageous in analyzing the number of interacting molecules because the probability of meeting at the same location is extremely low.
따라서, 살아있는 단일 세포에서 세포막 단백질들 간의 일시적인 상호작용을 정량적으로 분석할 수 있는 방법의 개발이 여전히 요구되고 있다.Therefore, there is still a need for development of a method capable of quantitatively analyzing transient interactions between membrane proteins in living single cells.
[선행기술문헌][Preceding technical literature]
[특허문헌][Patent Documents]
(특허문헌 1) 대한민국 특허출원 제10-2014-0059027호(Patent Document 1) Korean Patent Application No. 10-2014-0059027
(특허문헌 2) 대한민국 특허출원 제10-2015-0060619호(Patent Document 2) Korean Patent Application No. 10-2015-0060619
본 발명은 살아있는 단일 세포에서 세포막 단백질들 사이의 일시적인 상호작용을 정량적으로 분석할 수 있는 방법을 제공하고자 한다. The present invention seeks to provide a method for the quantitative analysis of transient interactions between membrane proteins in living single cells.
보다 구체적으로, 본 발명은 More specifically, the present invention
살아있는 세포에서 표적 막 단백질과 상기 표적 막 단백질에 대한 결합 파트너 막 단백질 간의 상호작용을 분석하는 방법에 있어서,A method for analyzing the interaction between a target membrane protein and a binding partner membrane protein for the target membrane protein in living cells,
분석 대상 세포를 기재 상에 위치시키고,Place the cell of interest on the substrate,
기재와 상기 표적 막 단백질 양자에 대한 결합능을 갖는 캡처 프로브를 분석 대상 세포에 처리하여 상기 표적 막 단백질을 기재 상에 고정시키고, A capture probe having binding capacity to both the substrate and the target membrane protein is treated with the cells of analysis to immobilize the target membrane protein on the substrate,
상기 캡처 프로브의 처리 전과 후의 상기 결합 파트너 막 단백질의 확산계수 분포를 얻는 것을 포함하는Obtaining diffusion coefficient distribution of the binding partner membrane protein before and after treatment of the capture probe.
막 단백질의 상호작용 분석 방법을 제공한다.Provided are methods for analyzing the interaction of membrane proteins.
본 발명에 있어서, 「분석 대상 세포」는 특정 막 단백질과 이와 상호작용할 수 있는 막 단백질 간의 상호작용을 분석하기 위해 사용되는 세포를 의미한다. 본 발명에 있어서 분석 대상 세포는 살아있는 세포이며, 그 종류는 어떠한 것이라도 관계없다. In the present invention, "cell to be analyzed" means a cell used for analyzing the interaction between a specific membrane protein and a membrane protein that can interact with it. In the present invention, the cells to be analyzed are living cells, and any kind thereof may be used.
한 구체예에서, 분석 대상 세포는 표적 막 단백질을 선택적으로 과발현시킨 세포일 수 있다. 표적 막 단백질의 과발현은 본 발명의 분석 방법에 있어서 필수적인 것은 아니다.In one embodiment, the cell under analysis can be a cell that selectively overexpresses the target membrane protein. Overexpression of the target membrane protein is not essential for the assay method of the present invention.
본 발명은 살아있는 세포 상에서 표적 막 단백질과 상기 표적 막 단백질에 대한 결합 파트너 막 단백질 간의 일시적인 상호작용을 정량적으로 분석하는 것을 목적으로 한다. The present invention aims to quantitatively analyze the transient interaction between a target membrane protein and a binding partner membrane protein for the target membrane protein on living cells.
본 발명에 있어서, 「표적 막 단백질」은 세포막에 위치하는 막 단백질로서, 다른 막 단백질과의 상호작용을 파악하길 원하는 막 단백질을 의미하며, 그 종류는 특별히 제한되지 않는다. 본 명세서의 하기 실시예에서는 「표적 막 단백질」을 「미끼 단백질(Bait)」로도 표현한다. 본 명세서에 있어서, 「표적 막 단백질」과 「미끼 단백질(Bait)」은 상호교환적으로 사용된다. 하기 실시예에서는 EGFR을 표적 막 단백질로 하여 이와 상호작용하는 막 단백질의 움직임 변화를 파악하였으나, EGFR 외의 막 단백질들도 표적 막 단백질로서 분석할 수 있다. In the present invention, the "target membrane protein" is a membrane protein located on the cell membrane, and means a membrane protein which wants to grasp the interaction with other membrane proteins, and the kind thereof is not particularly limited. In the following examples of the present specification, "target membrane protein" is also expressed as "bait protein". In this specification, "target membrane protein" and "bait protein" (Bait) are used interchangeably. In the following examples, EGFR is used as the target membrane protein, and the change in the movement of the membrane protein interacts with it. However, membrane proteins other than EGFR can also be analyzed as the target membrane protein.
본 발명에 있어서, 「결합 파트너 막 단백질」은 표적 막 단백질과 상호작용을 하는 단백질을 의미한다. 표적 막 단백질과의 상호작용을 하는 것으로 알려져 있거나 표적 막 단백질과 상호작용을 할 것으로 예상되는 막 단백질을 결합 파트너 막 단백질로서 설정하고 표적 막 단백질과의 상호작용을 분석할 수 있다. 본 명세서의 하기 실시예에서는 「결합 파트너 막 단백질」을 「먹이 단백질(Prey)」로도 표현한다. 본 명세서에 있어서, 「결합 파트너 막 단백질」과 「먹이 단백질(Prey)」은 상호교환적으로 사용된다.In the present invention, "binding partner membrane protein" means a protein that interacts with a target membrane protein. Membrane proteins known to or expected to interact with the target membrane protein can be set as binding partner membrane proteins and analyzed for interaction with the target membrane protein. In the following examples of the present specification, the "binding partner membrane protein" is also expressed as "prey protein" (Prey). In this specification, "binding partner membrane protein" and "feed protein (Prey)" are used interchangeably.
본 발명에 있어서, 「표적 막 단백질」과 표적 막 단백질에 대한 「결합 파트너 막 단백질」은 내재성 막 단백질(integral membrane protein), 표재성 막 단백질(peripheral membrane protein), 막관통 단백질(transmembrane protein), 막 당단백질(membrane glycoprotein) 및 지질 공정 막 단백질(lipid anchored membrane protein)으로 이루어진 군으로부터 선택되는 것일 수 있다. In the present invention, the "target membrane protein" and the "binding partner membrane protein" for the target membrane protein include an integral membrane protein, a peripheral membrane protein, a transmembrane protein, It may be selected from the group consisting of membrane glycoprotein (membrane glycoprotein) and lipid anchored membrane protein (lipid anchored membrane protein).
본 발명에 있어서, 「표적 막 단백질」과 상기 표적 막 단백질에 대한 「결합 파트너 막 단백질」은 막 단백질의 세포막 내 이동 양상을 추적하기 위해 「검출가능한 모이어티(detectable moiety) 」에 의해 레이블링될 수 있다. 「검출가능한 모이어티」에 의해 「표적 막 단백질」과 상기 표적 막 단백질에 대한 「결합 파트너 막 단백질」의 존재, 위치, 양 등을 파악할 수 있게 된다. 막 단백질을 추적하기 위한 검출가능한 모이어티는 당해 기술 분야에 잘 알려져 있으며, 예를 들어, 형광(fluorescent), 발광(luminescent), 화학발광(chemiluminescent), 방사성(radioactive) 모이어티일 수 있다. In the present invention, the "target membrane protein" and the "binding partner membrane protein" for the target membrane protein may be labeled by a "detectable moiety" to track the movement of the membrane protein in the cell membrane. have. The "detectable moiety" makes it possible to grasp the presence, the position, the amount of the "target membrane protein" and the "binding partner membrane protein" with respect to the target membrane protein. Detectable moieties for tracking membrane proteins are well known in the art and may be, for example, fluorescent, luminescent, chemiluminescent, radioactive moieties.
한 구체예에서, 막 단백질은 검출가능한 모이어티로서 형광 모이어티에 의해 레이블링되어 있는 것일 수 있다. 예를 들어, 막 단백질은 형광 단백질과의 융합 단백질의 형태로 발현시켜 탐지하기 용이하도록 제공될 수 있다. 다르게는 막 단백질은 유기형광염료가 결합될 수 있는 태그와의 융합 단백질이면서 상기 융합 단백질의 태그에 형광물질이 레이블링되어 있는 상태일 수 있다. 상기 유기형광염료가 결합될 수 있는 태그로는 SNAP tag나 CLIP tag을 들 수 있다. 예를 들어 SNAP tag나 CLIP tag를 막 단백질에 발현시키고, 이들 태그에 결합하는 벤질구아니딘 유도체 또는 O2-벤질사이토신 유도체의 말단에 형광물질을 결합시킴으로써 막 단백질에 형광모이어티를 레이블링시킨다. 또 다른 예로, 막 단백질에 특이적으로 결합하는 프로브에 의해 레이블링되어 있고, 여기에 다시 형광물질이 결합되는 형태로 형광 모이어티에 의한 레이블링이 구현될 수도 있다. 본 발명의 하기 실시예에서는 EGRF의 C 말단에 mEos3.2 단백질이 결합되어 있는 EGFR-mEos3.2로 발현시키거나, EGFR의 N 말단에 SNAP tag를 결합시켜 CF660R 형광염료로 표지한 EGFR을 표적 막 단백질 및/또는 결합 파트너 막 단백질의 레이블링에 사용한 예를 보여준다. 다르게는 막 단백질은 탐지가능한 모이어티에 의해 레이블링되어 있는 항체와의 바인딩을 통해 레이블링될 수도 있다.In one embodiment, the membrane protein may be one labeled by the fluorescent moiety as a detectable moiety. For example, membrane proteins may be provided to facilitate expression by expression in the form of fusion proteins with fluorescent proteins. Alternatively, the membrane protein may be a fusion protein with a tag to which an organic fluorescent dye may be bound, and a fluorescent material is labeled on a tag of the fusion protein. Examples of tags to which the organic fluorescent dyes may be combined include SNAP tags and CLIP tags. For example, a fluorescent moiety is labeled on a membrane protein by expressing a SNAP tag or a CLIP tag on a membrane protein and binding a fluorescent substance to a terminal of a benzylguanidine derivative or O2-benzylcytosine derivative that binds to these tags. As another example, labeling by a probe that specifically binds to a membrane protein may be implemented by a fluorescent moiety in a form in which a fluorescent material is bound thereto. In the following example of the present invention, EGFR labeled with EGFR-mEos3.2 having the mEos3.2 protein bound to the C terminus of EGRF, or bound with the SNAP tag to the N terminus of EGFR, is labeled with a target membrane of EGFR. Examples used for labeling proteins and / or binding partner membrane proteins are shown. Alternatively the membrane protein may be labeled via binding to an antibody labeled by a detectable moiety.
한 구체예에서, 막 단백질과 융합되는 상기 형광 단백질은 GFP(Green Fluorescent Protein) 계열, BFP(Blue fluorescent protein) 계열, Cyan 계열, YFP(Yellow Fluorescent Protein) 계열, RFP(Red Fluorescent Protein) 계열, Orange 계열, Far-red 계열, Near-IR, 광활성 단백질(Photoactivatable protein), 광전환 단백질(Photoconvertible protein), 및 광스위치 단백질(Photoswitchable protein)로 이루어진 군으로부터 1종 이상 선택되는 것일 수 있다. 예를 들어, 상기 형광 단백질은 EGFP(enhanced green fluorescent protein), Emerald, Superfolder GFP, Azami green mWasabi, TagGFP, AcGFP, T-sapphire, mUKG, Clover, mNeonGreen, EBFP(enhanced blue fluorescent protein), EBFP2, Azurite, mTagBFP, mKalama1, Sirius, ECFP(enhanced cyan fluorescent protein), mECFP(monomeric ECFP), Cerulean, mTurquoise, mTurquoise2, CyPet, TagCFP, mTFP1(Teal), SCFP3A, monomeric Midoriishi Cyan, EYFP(enhanced yellow fluorescent protein), Topaz, Benus, mCitrine, YPet, TagYFP, PhiYFP, mBanana, SYFP2, mRuby, mRuby2, mApple, mStrawberry, mRFP1, mCherry, mRaspberry, dKeima-Tandem(monomeric version), HcRed-Tandem(monomeric version), mPlum, mKate2, mNeptune, mKate2, mNeptune, TagRFP657, IFP1.4, PA-GFP, PAmCherry1, PaTagRFP, PS-CFP2, mEos2, mEos3.2, PSmOrange 및 Dronpa로 이루어진 군으로부터 1종 이상 선택되는 것일 수 있다.In one embodiment, the fluorescent protein is fused with a membrane protein is Green Fluorescent Protein (GFP) series, Blue fluorescent protein (BFP) series, Cyan series, Yellow Fluorescent Protein (YFP) series, Red Fluorescent Protein (RFP) series, Orange It may be one or more selected from the group consisting of series, Far-red series, Near-IR, photoactivatable protein, photoconvertible protein, and photoswitchable protein. For example, the fluorescent protein is enhanced green fluorescent protein (EGFP), Emerald, Superfolder GFP, Azami green mWasabi, TagGFP, AcGFP, T-sapphire, mUKG, Clover, mNeonGreen, enhanced blue fluorescent protein (EBFP), EBFP2, Azurite , mTagBFP, mKalama1, Sirius, enhanced cyan fluorescent protein (ECFP), monomeric ECFP (mECFP), Cerulean, mTurquoise, mTurquoise2, CyPet, TagCFP, mTFP1 (Teal), SCFP3A, monomeric Midoriishi Cyan, EYFP (enhanced yellow fluorescent protein) Topaz, Benus, mCitrine, YPet, TagYFP, PhiYFP, mBanana, SYFP2, mRuby, mRuby2, mApple, mStrawberry, mRFP1, mCherry, mRaspberry, dKeima-Tandem (monomeric version), HcRed-Tandem (monomeric version) mNeptune, mKate2, mNeptune, TagRFP657, IFP1.4, PA-GFP, PAmCherry1, PaTagRFP, PS-CFP2, mEos2, mEos3.2, PSmOrange and Dronpa may be one or more selected from the group consisting of.
막 단백질과 형광단백질의 융합 단백질은 막 단백질과 형광단백질의 유전적 융합이나 화학결합으로 형성된 공유결합 복합체를 의미한다. 여기에서, “유전적 융합”이란 단백질을 코딩하는 DNA 서열의 유전적 발현을 통해서 형성된 선형, 공유결합으로 이루어진 연결을 의미한다. 융합 단백질의 제조는 공지된 방법으로 수행될 수 있으며, 예컨대, 유전자 재조합 기술을 이용하여 융합 단백질을 제조할 수 있다.Fusion protein of membrane protein and fluorescent protein refers to a covalent complex formed by genetic fusion or chemical bonding of membrane protein and fluorescent protein. Here, "genetic fusion" means a linear, covalent linkage formed through the genetic expression of a DNA sequence encoding a protein. The preparation of the fusion protein can be carried out by known methods, for example, the fusion protein can be prepared using genetic recombination techniques.
상기 융합 단백질을 발현하는 세포는 발현벡터를 형질전환시켜 준비할 수 있다. 융합 단백질의 발현을 위해 개발된 공지의 발현벡터를 적절히 선택하여 사용할 수 있다. 예컨대, 상기 발현벡터는 플라스미드 벡터, 바이러스 또는 코스미드 벡터일 수 있다. 융합 단백질을 발현하는 숙주세포는 문헌(Sambrook, J., et al.,Molecular Cloning, A Laboratory Manual(2판), Cold Spring Harbor Laboratory, 1. 74, 1989)에 기재된 인산칼슘법 또는 염화칼슘/염화루비듐법, 일렉트로포레이션법(electroporation), 전기주입법(electroinjection), PEG 등의 화학적 처리방법, 유전자총(gene gun)을 이용하는 방법 및 바이러스 트랜스펙션(virus transfection) 등의 형질전환방법을 이용하여 준비될 수 있다. 예컨대, 표적 막 단백질과 형광단백질의 융합 단백질이 포함된 발현벡터를 리포펙타민(lipofectamine), 퓨진 (fugene), 또는 메타팩틴(metafectin)을 이용하여 숙주세포에 형질전환시켜 융합 단백질을 발현하는 세포를 준비할 수 있다.Cells expressing the fusion protein can be prepared by transforming the expression vector. Known expression vectors developed for the expression of the fusion protein can be appropriately selected and used. For example, the expression vector may be a plasmid vector, a virus or a cosmid vector. Host cells expressing the fusion protein are either calcium phosphate method or calcium chloride / chloride described in Sambrook, J., et al., Molecular Cloning, A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory, 1. 74, 1989. Rubidium, electroporation, electroinjection, chemical treatment such as PEG, gene gun, and transfection such as virus transfection Can be prepared. For example, a cell expressing a fusion protein by transforming an expression vector including a fusion protein of a target membrane protein and a fluorescent protein into a host cell using lipofectamine, fugene, or metafectin. You can prepare.
한편, 상기 형광물질은 유기형광염료일 수 있으며, 그 종류는 특별히 제한되지 않는다. 예를 들어, 유기형광염료는 CF660R, Alexa Flour 647, Atto 488, Alexa Flour 488, Dy505, Rhodamine 123, Atto 520, Dy 530, ATTO 532, Alexa Fluor 532, Fluorescein, FITC, Cy2, Cy3B, Alexa Flour 568, TAMRA, Cy3, Cy3.5, SNAP-Cell TMR-Star, Atto 565, Atto 590, Alexa Fluor 647, Cy5, Atto 647, Atto 647N, Dyomics 654, Atto 655, TMP-ATTO 655, Atto 680, Cy5.5, Atto 680, Alexa Fluor 680, Atto 700, Alexa Fluor 700, DyLight 750, Cy7, Alexa Flour 750, Atto 740, Alexa Flour 790, silicon-rhodamine (SiR) 또는 IRDye 800 CW 등과 같은 공지의 유기형광염료로부터 당업자가 적절히 선택할 수 있다. On the other hand, the fluorescent material may be an organic fluorescent dye, the kind is not particularly limited. For example, organic fluorescent dyes are CF660R, Alexa Flour 647, Atto 488, Alexa Flour 488, Dy505, Rhodamine 123, Atto 520, Dy 530, ATTO 532, Alexa Fluor 532, Fluorescein, FITC, Cy2, Cy3B, Alexa Flour 568 , TAMRA, Cy3, Cy3.5, SNAP-Cell TMR-Star, Atto 565, Atto 590, Alexa Fluor 647, Cy5, Atto 647, Atto 647N, Dyomics 654, Atto 655, TMP-ATTO 655, Atto 680, Cy5. 5, from known organic fluorescent dyes such as Atto 680, Alexa Fluor 680, Atto 700, Alexa Fluor 700, DyLight 750, Cy7, Alexa Flour 750, Atto 740, Alexa Flour 790, silicon-rhodamine (SiR) or IRDye 800 CW One skilled in the art can make appropriate selections.
본 발명에 있어서, 「기재」는 분석 대상 세포의 막 단백질을 고정화시키고 막 단백질의 고정 및 확산 양상을 관찰하기 위해 사용되는 플레이트, 웰 등을 의미한다. 본 발명의 한 구체예에서, 기재는 슬라이드 글라스 또는 커버글라스일 수 있다. 본 발명에 따른 기재는 소재의 제한은 없으나, 막 단백질의 움직임을 추적할 수 있도록 레이블링되는 형광물질 등의 검출가능한 모이어티를 검출하기에 용이한 것이 좋다.In the present invention, "substrate" means a plate, a well, or the like used for immobilizing the membrane protein of the cell to be analyzed and observing the immobilization and diffusion of the membrane protein. In one embodiment of the invention, the substrate may be a slide glass or cover glass. The substrate according to the present invention is not limited to the material, but it is preferable to easily detect a detectable moiety such as a fluorescent substance labeled to track the movement of the membrane protein.
본 발명에 있어서, 「캡처 프로브」는 표적 막 단백질을 기재 상에 고정하기 위해 사용되는 프로브를 의미한다. 한 구체예에서, 상기 캡처 프로브는 항체, 항원-결합 단편, 어피바디(affibody), 또는 앱타머일 수 있다. In the present invention, "capture probe" means a probe used to immobilize a target membrane protein on a substrate. In one embodiment, the capture probe can be an antibody, antigen-binding fragment, affibody, or aptamer.
캡처 프로브는 막 단백질과 기재 간의 적절한 접촉이 일어날 수 있는 높이로 제공될 수 있다. 예를 들어, 캡처 프로브가 항체일 때 기재와 세포 간의 접촉이 잘 일어나지 않는 경우 상기 캡처 프로브로 사용되는 항체에 적절한 높이를 제공해 줄 수 있는 2차 항체를 추가적으로 사용할 수 있을 것이다. The capture probe can be provided at a height such that proper contact between the membrane protein and the substrate can occur. For example, when contact between the substrate and the cell is difficult to occur when the capture probe is an antibody, a secondary antibody may be additionally used to provide an appropriate height to the antibody used as the capture probe.
본 발명에 따른 캡처 프로브는 기재와 상기 표적 막 단백질 양자에 대한 결합능을 갖는다. 이는 캡처 프로브의 처리 전과 후의 결합 파트너 막 단백질의 확산계수 분포를 얻기 위해서인데, 예를 들어, 아비딘-비오틴 결합법(avidin-biotin complex technique)를 이용하면 캡처 프로브가 기재와 상기 표적 막 단백질 양자에 대한 결합능을 갖도록 설계하기가 용이하다. 예를 들어, 기재 상에 아비딘을 결합시키고, 캡처 프로브에 비오틴을 컨쥬게이션하는 방법을 채택하게 되면, 아비딘이 결합된 기재만을 준비해 두면, 막 단백질의 종류의 변경에 따라 비오틴이 컨쥬게이션된 캡처 프로브의 종류만 변경하여 본 발명의 방법을 수행하면 된다. 본 발명의 구체예에서, 기재는 뉴트라비딘, 스트렙타비딘, 또는 아비딘이 결합되어 있는 것일 수 있다. 뉴트라비딘, 스트렙타비딘, 또는 아비딘을 공유결합을 통해 기재 상에 결합시키는 방법은 당업계에 잘 알려져 있다. Capture probes according to the invention have the ability to bind both the substrate and the target membrane protein. This is to obtain the diffusion coefficient distribution of binding partner membrane proteins before and after the treatment of the capture probes. For example, using the avidin-biotin complex technique, capture probes can be applied to both the substrate and the target membrane protein. It is easy to design to have a binding capacity for. For example, when adopting a method of binding avidin on a substrate and conjugating biotin to a capture probe, if only the substrate to which avidin is bound is prepared, a capture probe in which biotin is conjugated according to a change in the type of membrane protein It is only necessary to change the type of to perform the method of the present invention. In an embodiment of the invention, the substrate may be a neutravidin, streptavidin, or avidin is bound. Methods of binding neutravidin, streptavidin, or avidin on a substrate via covalent bonds are well known in the art.
용어 「확산계수(diffusion coefficient)」는 확산하는 분자의 평균속도를 의미하는데, 본 발명에서는 막 단백질의 세포막 내 이동성을 나타내는 지표이다. The term "diffusion coefficient" refers to the average velocity of molecules to diffuse, and in the present invention is an index indicating the mobility of membrane proteins in the cell membrane.
한 구체예에서, 막 단백질의 확산계수는 단일 입자 추적(single particle tracking, SPT)을 통해 막 단백질의 세포막 내 이동궤적을 탐지함으로써 얻을 수 있다. 단일 입자 추적이란 어떠한 medium 안에서 단일 입자의 움직임을 관찰하는 것으로 시간에 따른 움직임의 좌표를 궤도(trajectory)로 표현하며, 이로부터 움직임 양식, 불균질성 등에 대해서 분석하는 기술이다(Saxton, M. J., Jacobson, K. Single-particle tracking: Applications to membrane dynamics. Annual Review of Biophysics and Biomolecular Structure 1997;26(373-399). In one embodiment, the diffusion coefficient of the membrane protein can be obtained by detecting the trajectory in the cell membrane of the membrane protein through single particle tracking (SPT). Single particle tracking is a technique of observing the movement of a single particle in a medium and expressing the coordinates of the movement over time as a trajectory, and analyzing the movement pattern and heterogeneity (Saxton, MJ, Jacobson, K). Single-particle tracking: Applications to membrane dynamics.Annual Review of Biophysics and Biomolecular Structure 1997; 26 (373-399).
막 단백질의 세포막 내 이동 궤적의 탐지는 당해 기술분야에서 공지된 방법으로 수행될 수 있다. 예컨대, TIRF(total internal reflection fluorescence)를 이용하여 형광을 탐지할 수 있다. TIRF 현미경은 전반사 현미경으로서 빛의 전반사 현상을 이용하여 다른 빛에 의한 간섭을 배제하고 특정 200nm 이하 영역의 형광만을 관찰할 수 있기 때문에 세포막과 그 주변의 현상을 연구하는데 적합하다. 이러한 TIRF 현미경을 이용하여 막 단백질의 시간에 따른 위치에서의 이미지를 얻고, 이를 궤도로 표현하여 확산계수를 구하게 된다.Detection of migration trajectories in the cell membrane of membrane proteins can be carried out by methods known in the art. For example, fluorescence may be detected using total internal reflection fluorescence (TIRF). TIRF microscope is a total reflection microscope, which is suitable for studying the cell membrane and its surroundings because it is possible to observe the fluorescence in a specific region below 200 nm by excluding the interference by other light by using the total reflection phenomenon of light. The TIRF microscope is used to obtain an image of a membrane protein at a time-dependent position and to express it in orbit to obtain a diffusion coefficient.
막 단백질의 확산계수는 하기 [수학식 1] 및 [수학식 2]를 이용하여 구할 수 있다. 예를 들어, 결합 파트너 막 단백질 단일 입자의 확산계수는 단일 입자 추적 분석을 통해 결합 파트너 막 단백질 단일 입자의 궤적을 얻고, 상기 [수학식 1] 및 [수학식 2]를 통해 구할 수 있게 된다. The diffusion coefficient of the membrane protein can be calculated using the following Equation 1 and Equation 2. For example, the diffusion coefficient of a single binding partner membrane protein single particle can be obtained through the single particle tracking analysis to obtain the locus of the binding partner membrane protein single particle, and can be obtained through Equation 1 and Equation 2.
막 단백질 단일 입자의 궤적은 단일 입자 추적 분석을 통해 시간에 따른 막 단백질의 위치 변화를 보여주는 복수 개의 막 단백질 이미지를 얻고, 상기 이미지로부터 막 단백질의 위치를 탐지하고, 탐지된 위치들을 연결하여 얻어진다. 예컨대, 막 단백질에 연결된 형광단백질의 형광이나 유기형광염료의 형광을 탐지하여 시간에 따른 막 단백질의 위치, 즉 좌표를 소정의 시간 동안 여러 개의 이미지로 얻고, 이러한 좌표의 움직임을 하나의 표적 막 단백질의 궤적으로 표현할 수 있다. 그런 다음, 하기 수학식 1 및 2에 측정된 시간에 따른 궤적의 좌표들을 대입하여 하나의 궤적에 대한 확산계수를 구할 수 있다. 위와 같은 계산에서 하나의 막 단백질 입자의 위치를 결정하기 위해 형광을 탐지하는 시간(한 좌표에서의 막 단백질 입자를 이미징하는데 걸리는 시간)은 분석 조건에 따라 적절히 변경할 수 있는데, 예를 들어, 약 30 내지 70 ms, 예컨대, 50 ms일 수 있다. 또한, 하나의 표적 막 단백질 입자가 하나의 궤적을 만들 때 필요한 좌표는 이에 제한되는 것은 아니나 5개 이상인 것이 바람직하다. 이에 제한되는 것은 아니나, 단일 입자 추적 분석에 사용되는 막 단백질 단일 입자의 궤적의 수는 3천개 내지 1만개, 바람직하게는 5천개 이상의 궤적일 수 있다. 불확실성을 줄이면서 수렴된 확산 계수 값을 얻기 위한 궤적의 수는 세포의 종류나 상태 등에 따라 다를 수 있으므로 적합한 분석을 위한 궤적의 수는 상황에 따라 달리 조절할 수 있다.The trajectory of a membrane protein single particle is obtained by obtaining a plurality of membrane protein images showing the change of membrane protein position over time through single particle tracking analysis, detecting the position of the membrane protein from the image, and concatenating the detected positions. . For example, by detecting the fluorescence of the fluorescent protein linked to the membrane protein or the fluorescence of the organic fluorescent dye, the position of the membrane protein over time, i.e., coordinates, can be obtained as a plurality of images for a predetermined time, and the movement of the coordinates is one target membrane protein It can be expressed as the trajectory of. Then, by substituting the coordinates of the trajectory with time measured in Equations 1 and 2, a diffusion coefficient for one trajectory may be obtained. In this calculation, the time for detecting fluorescence (the time taken to image the membrane protein particles at one coordinate) to determine the position of one membrane protein particle can be changed according to the analysis conditions, for example, about 30 To 70 ms, for example 50 ms. In addition, the coordinates required when one target membrane protein particle makes one trajectory is not limited thereto, but preferably five or more. Although not limited thereto, the number of trajectories of the membrane protein single particles used in the single particle trace analysis may be from 3000 to 10,000, preferably 5,000 or more. The number of trajectories for obtaining convergent diffusion coefficient values while reducing uncertainty may vary depending on cell type or condition, so the number of trajectories for appropriate analysis can be adjusted according to the situation.
[수학식 1][Equation 1]
Figure PCTKR2017005544-appb-I000001
Figure PCTKR2017005544-appb-I000001
(MSD: 이동한 거리 제곱 평균(Mean Square Displacement), Δ: 표적 막 단백질 입자의 좌표 간 지연 시간, T: 하나의 궤적을 이루는 표적 막 단백질 입자의 좌표의 총 수, (x0, y0): 시작 위치, (xt,yt): 시간 t에서의 위치)(MSD: Mean Square Displacement, Δ: delay time between coordinates of target membrane protein particles, T: total number of coordinates of target membrane protein particles forming one trajectory, (x 0 , y 0 ) : Start position, (x t , y t ): position at time t)
[수학식 2][Equation 2]
MSD(Δ) = 4DΔ MSD (Δ) = 4DΔ
(D: 확산계수,Δ: 표적 막 단백질 입자의 좌표 간 지연 시간)(D: diffusion coefficient, Δ: delay time between coordinates of the target membrane protein particles)
위와 같은 계산에서 MSD는 표적 막 단백질 입자의 좌표 간 스텝 크기에 따른 함수로 나타나며 해당 궤적을 브라운 운동에 의한 것으로 가정하였을 때 MSD는 선형함수로 나타난다. 따라서 [수학식 2]에 의해 각 막 단백질의 궤적의 MSD를 선형함수 (f(X)=aX)에 fit 하였을 때의 기울기로부터 그 궤적의 확산계수 (a/4)를 얻을 수 있다. MSD는 평균값을 나타내기 때문에 해당 궤적을 이루는 막 단백질 입자의 좌표가 많을수록 (즉, 추적하는 궤적이 길어지거나 좌표 밀도가 높아질수록) 더 정확한 확산계수를 얻을 수 있다. 따라서 확산계수의 계산은 일정한 길이 이상의 궤적을 사용하며 선형함수의 fit은 표적 막 단백질 입자의 좌표 간 스텝 크기를 두 프레임 이상으로 하여 (선형함수의 fit은 두 점이 있으면 가능하기 때문에) 확산계수의 측정의 정확도를 높인다. 이러한 확산계수를 전체 궤적에 대해 평균하여 각 세포 내의 막 단백질의 평균 확산계수를 계산할 수 있다.In the above calculation, the MSD is represented as a function of the step size between the coordinates of the target membrane protein particles, and the MSD is represented as a linear function when the trajectory is assumed by Brownian motion. Therefore, the diffusion coefficient (a / 4) of the trajectory can be obtained from the slope when the MSD of the trajectory of each membrane protein is fit to the linear function (f (X) = aX) by [Equation 2]. Since the MSD represents an average value, the more the coordinates of the membrane protein particles forming the trajectory (ie, the longer the tracking trajectory or the higher the coordinate density), the more accurate the diffusion coefficient. Therefore, the calculation of the diffusion coefficient uses a trajectory over a certain length, and the fit of the linear function measures the diffusion coefficient with the step size between the coordinates of the target membrane protein particles more than two frames (since the fit of the linear function is possible with two points). To increase the accuracy. These diffusion coefficients can be averaged over the entire trajectory to calculate the average diffusion coefficient of the membrane protein in each cell.
다른 구체예에서, 상기 결합 파트너 막 단백질의 확산계수는 FRAP(Fluorescence recovery after photobleaching) 또는 FCS(Fluorescence Correlation Spectroscopy)에 의해 얻을 수 있다. FRAP은 세포막에서 막 단백질의 측방 확산을 측정하는 방법으로 잘 알려져 있다. 표적 막 단백질을 형광 표지한 후 세포막에 도입하여 세포의 특정 부분에 레이저광을 조사하여 형광을 퇴색시킨 후, 주위에서 표적 막 단백질의 측방확산에 의해 형광이 회복되는 상태를 측정하는 방법이다. FRAP에서 표적 막 단백질의 확산 계수 D는 D = (w 2/4τ 1 / 2)γ에서 산출한다. 여기서 w는 레이저 광속 폭으로 광속이 정규(가우스) 분포를 하는 것으로 가정할 때, 중심부 밝기가 e-2로 감쇠하는 거리에서 τ 1 /2는 형광이 반 분가량 회복하는 시간이고, γ는 형광 퇴색 강도에 의존하는 보정치가 된다. FCS는 시간에 따른 형광 세기의 요동을 측정하고, 자기 상관(auto-correlation)을 분석함으로써 액체 안에서 움직이는 형광체의 확산계수를 구할 수 있다. 매우 작은 초점 공간에서 광학적으로 관찰하고 있는 형광의 세기는 평균값을 중심으로 시간에 따라 무작위하게 변화하는데, 이 무작위한 요동은 형광체가 브라운 운동(Brownian motion)을 함에 따라 초점 공간에 들어왔다 나갔다 하는 정보를 담고 있다. 따라서, 확산계수가 낮을수록 초점 공간 안에 들어왔다 나갔다 하는 속도가 느리기 때문에 자기 상관 시간이 느려지게 된다. FCS를 통한 자기 상관 G(τ)은 G(τ)=[G(0)/{(1+(τ/τD))*(1+a- 2(τ/τD))1 /2)}]+G(∞)로 나타난다. 여기서 a는 wz/wxy로 초점 공간 부피에 대한 초점 평면과 그 수직 축의 반지름에 대한 비율이고, 각각의 광학 현미경의 상태에 따른 고유 수치로 미리 결정이 되어있다. 따라서, 자기 상관을 측정함으로써 τD를 결정할 수 있다. 이 때 형광체의 확산 계수 D는 D=wxy 2/4τD로 산출된다.In another embodiment, the diffusion coefficient of the binding partner membrane protein may be obtained by Fluorescence recovery after photobleaching (FRAP) or Fluorescence Correlation Spectroscopy (FCS). FRAP is well known for measuring lateral diffusion of membrane proteins in cell membranes. After fluorescently labeling a target membrane protein, it is introduced into a cell membrane to irradiate a specific portion of the cell with laser light to fluoresce the fluorescence, and then measure a state in which fluorescence is recovered by lateral diffusion of the target membrane protein around. Diffusion coefficient D of the target membrane protein in FRAP is calculated at D = (w 2/4 τ 1/2) γ. Where w is, assuming that the light beam with a laser beam width of a normal (Gaussian) distribution, and the time in the distance to the center of brightness attenuation to e-2 τ 1/2 is the fluorescence recovery for about a half minute, γ Fluorescence The correction value depends on the fading intensity. FCS can measure the diffusion coefficient of fluorescence in a liquid by measuring fluctuations in fluorescence intensity over time and analyzing auto-correlation. The intensity of fluorescence optically observed in a very small focal space varies randomly with time around the mean value, and this random fluctuation shows information that the phosphor enters and exits the focal space as Brownian motion occurs. It contains. Therefore, the lower the diffusion coefficient is, the slower the autocorrelation time is due to the slower speed of entering and exiting the focal space. Auto-correlation G (τ) through the FCS is G (τ) = [G ( 0) / {(1+ (τ / τ D)) * (1 + a - 2 (τ / τ D)) 1/2) }] + G (∞). Where a is the ratio of the focal plane to the focal plane volume and the radius of its vertical axis at w z / w xy , and is determined in advance by intrinsic values according to the state of each optical microscope. Therefore, τ D can be determined by measuring autocorrelation. At this time, the diffusion coefficient D of the phosphor is calculated as D = w xy 2 / 4τ D.
본 발명의 분석 방법에 따라, 캡처 프로브를 분석 대상 세포에 처리하면 기재 상에 표적 막 단백질이 고정되게 되는데, 만일 캡처 프로브의 처리 후 결합 파트너 막 단백질의 이동성, 즉 결합 파트너 막 단백질의 확산계수가 변화하게 되면, 이는 결합 파트너 막 단백질 부분모집단의 확산계수 분포의 변화로 나타나게 된다. 확산계수 분포도라는 것은 이런 단일 분자 수준의 확산계수들의 히스토그램을 의미하는데, 가로축은 확산계수를, 세로축은 해당 확산계수를 갖는 단분자의 개수 또는 표준화 이후에는 전체 대비 해당 단분자의 비율을 나타낸다. 분포도의 불확실성과 오차 범위를 줄이고 가우시안 그래프로서 피팅하기 위해서는 1,000개 이상의 단분자 확산계수가 측정되는 것이 유리할 수 있으나, 이에 제한되는 것은 아니다.According to the analytical method of the present invention, when the capture probe is treated to the cells to be analyzed, the target membrane protein is immobilized on the substrate. If the capture probe is treated, the mobility of the binding partner membrane protein, that is, the diffusion coefficient of the binding partner membrane protein, is fixed. When changed, this results in a change in the diffusion coefficient distribution of the binding partner membrane protein subpopulation. Diffusion coefficient distribution means a histogram of diffusion coefficients at the level of a single molecule. The horizontal axis represents the diffusion coefficient and the vertical axis represents the number of single molecules having the corresponding diffusion coefficient or the ratio of the single molecules to the whole after standardization. In order to reduce the uncertainty and error range of the distribution and to fit the Gaussian graph, it may be advantageous to measure more than 1,000 single molecule diffusion coefficients, but is not limited thereto.
보다 구체적으로, 결합 파트너 막 단백질이 기재 상에 고정된 표적 막 단백질과 상호작용하는 경우, 단일 분자 수준에서 살펴볼 때 결합 파트너 막 단백질의 확산계수는 낮아지게 될 것이다. 그런데, 세포막에는 이질적(heterogenous)인 상태로 존재하는 다수의 표적 단백질과 다수의 결합 파트너 막 단백질이 혼재되어 있다. 다시 말해, 표적 막 단백질과 결합 파트너 막 단백질은 양자 모두 이질적 상태의 단일분자들의 모집단(population)의 형태로 존재하며 각 단일분자의 확산계수에 따라 부분모집단(subpopulation)으로 분류할 수 있다. 세포막에 존재하는 표적 막 단백질 모집단이 캡처 프로브에 의해 기재 상에 고정되면 캡처 프로브의 처리 전과 비교하였을 때 표적 막 단백질의 확산계수 분포는 변화를 겪게 되는데, 캡처 프로브에 의하여 직접적으로 기재 상에 고정화되는 과정에 의해서 확산계수가 높은 부분모집단은 사라지고, 확산계수가 낮은 부분 모집단의 수는 늘어나는 확산계수 분포의 변화를 관찰할 수 있다. More specifically, when the binding partner membrane protein interacts with the target membrane protein immobilized on the substrate, the diffusion coefficient of the binding partner membrane protein will be lower when viewed at the single molecule level. However, the cell membrane is a mixture of a plurality of target proteins and a plurality of binding partner membrane proteins that exist in a heterogeneous (heterogenous) state. In other words, the target membrane protein and the binding partner membrane protein both exist in the form of a population of single molecules in heterogeneous states and can be classified into subpopulations according to the diffusion coefficient of each single molecule. When the target membrane protein population present in the cell membrane is immobilized on the substrate by the capture probe, the diffusion coefficient distribution of the target membrane protein undergoes a change when compared to the pretreatment of the capture probe, which is directly immobilized on the substrate by the capture probe. As a result, it is possible to observe a change in the distribution of diffusion coefficients, in which the subpopulations with high diffusion coefficients disappear and the number of subpopulations with low diffusion coefficients increases.
마찬가지로, 기재 상에 고정된 표적 막 단백질의 부분모집단과 상호작용하는 결합 파트너 막 단백질은 세포막 상에 자유롭게 돌아다니고 있던 결합 파트너 막 단백질 모집단 중 일부이며, 이러한 일부 결합 파트너 막 단백질을 결합 파트너 막 단백질의 부분모집단으로 일컫는다. 도 1에서 볼 수 있는 바와 같이, 캡처 프로브의 처리에 의해 표적 막 단백질이 기재 상에 고정화됨에 따라 이와 상호작용하고 있는 결합 파트너 막 단백질의 확산계수가 단일 분자 수준에서 감소하게 되며, 이는 확산계수가 높은 부분모집단의 수는 줄고, 확산계수가 낮은 부분모집단의 수가 증가하는 것으로 표현된다. 이러한 확산계수 분포의 변화는 결합 파트너 막 단백질이 표적 막 단백질과 상호작용하는 경향성이 클수록 더욱 크게 나타나게 된다.Similarly, binding partner membrane proteins that interact with a subset of target membrane proteins immobilized on a substrate are part of a population of binding partner membrane proteins that have been freely roaming on the cell membrane, and some of these binding partner membrane proteins It is called a subpopulation. As can be seen in Figure 1, as the target membrane protein is immobilized on the substrate by the treatment of the capture probe, the diffusion coefficient of the binding partner membrane protein interacting with it is reduced at the single molecule level, which is the diffusion coefficient The number of high subpopulations is reduced and the number of subpopulations with low diffusion coefficients is expressed. This change in diffusion coefficient distribution is more pronounced as the binding partner membrane protein tends to interact with the target membrane protein.
다시 말해, 상기 캡처 프로브의 처리 후의 상기 결합 파트너 막 단백질의 확산계수 분포에서 확산계수가 낮은 부분모집단의 수가 증가하는 것은 상기 표적 막 단백질과의 일시적인 상호작용, 즉, 복합체 형성이 일어났음을 의미한다.In other words, an increase in the number of subpopulations having a low diffusion coefficient in the diffusion coefficient distribution of the binding partner membrane protein after treatment of the capture probe means that a temporary interaction with the target membrane protein, that is, complex formation, has occurred. .
한편, 표적 막 단백질과 결합 파트너 막 단백질 간의 상호작용은 “일시적”인 것으로, 결합 파트너 단백질은 일시적으로 표적 막 단백질과 복합체를 형성하였다가 해리되게 되는데, 본 발명에 따른 막 단백질의 상호작용 분석 방법은 표적 막 단백질에 대한 결합 파트너 단백질의 해리상수를 구하는 것을 가능하게 해 준다.Meanwhile, the interaction between the target membrane protein and the binding partner membrane protein is "temporary", and the binding partner protein temporarily forms a complex with the target membrane protein and then dissociates. The method of analyzing the interaction of the membrane protein according to the present invention Makes it possible to find the dissociation constant of the binding partner protein for the target membrane protein.
한 구체예에서, 본 발명에 따른 막 단백질의 상호작용 분석 방법은 상기 캡처 프로브의 처리에 따른 확산계수 분포의 변화로부터 표적 막 단백질과 상기 표적 막 단백질에 대한 결합 파트너 막 단백질 간의 해리상수를 측정하는 것을 추가로 포함할 수 있다.In one embodiment, the method for analyzing the interaction of a membrane protein according to the present invention measures dissociation constant between a target membrane protein and a binding partner membrane protein for the target membrane protein from a change in diffusion coefficient distribution following treatment of the capture probe. It may further include.
「해리 상수」는 표적 막 단백질과 결합 파트너 막 단백질이 복합체를 형성하였다가 다시 단일 단백질로 해리되는 경향성을 의미한다. 표적 막 단백질과 상기 표적 막 단백질에 대한 결합 파트너 막 단백질 간의 해리상수는 하기 [수학식 3], [수학식 4] 또는 [수학식 5]를 통해 구할 수 있다. The "dissociation constant" refers to the tendency of the target membrane protein and the binding partner membrane protein to form a complex and then dissociate into a single protein. The dissociation constant between the target membrane protein and the binding partner membrane protein for the target membrane protein can be obtained by the following Equation 3, Equation 4 or Equation 5.
[수학식 3][Equation 3]
KD = [R][I] / [RI] K D = [R] [I] / [RI]
여기에서, [I], [R], [RI]는 각각 표적 막 단백질의 농도, 상기 표적 막 단백질에 대한 결합 파트너 막 단백질의 농도 및 표적 막 단백질과 상기 표적 막 단백질에 대한 결합 파트너 막 단백질 간의 복합체의 농도를 나타낸다.Wherein [I], [R], and [RI] are the concentrations of the target membrane protein, the concentration of the binding partner membrane protein for the target membrane protein, and between the target membrane protein and the binding partner membrane protein for the target membrane protein, respectively. The concentration of the complex is shown.
[수학식 4][Equation 4]
KD = [R]0(1-ρR)([I]0-[RI]) / [R]0ρR K D = [R] 0 (1-ρ R ) ([I] 0- [RI]) / [R] 0 ρ R
여기에서, [R]0와 [I]0, 그리고 ρR은 각각 상호작용이 발생하기 이전의 초기 표적 막 단백질 및 결합 파트너 막 단백질의 농도, 캡쳐 프로브 처리 전과 비교하여 처리 후에 증가된 결합 파트너 막 단백질의 부분모집단 비율이다.Wherein [R] 0 , [I] 0 , and ρ R are the concentrations of the initial target membrane protein and binding partner membrane protein before the interaction occurs, respectively, after binding probe membrane treatment, compared to before the capture probe treatment. The percentage of subpopulations of proteins.
[수학식 5] [Equation 5]
KD = (1-ρR)[I]0 / ρR K D = (1-ρ R ) [I] 0 / ρ R
여기에서, From here,
ρR은 캡처 프로브 처리 전과 비교하여 처리 후에 증가된 결합 파트너 막 단백질의 부분모집단 비율이고, [I0]는 고정화된 표적 막 단백질의 절대 농도를 나타낸다.ρ R is the subpopulation ratio of binding partner membrane protein increased after treatment compared to before capture probe treatment, and [I 0 ] represents the absolute concentration of immobilized target membrane protein.
일반적으로 해리 상수는 [수학식 3]과 같이 표적 막 단백질, 결합 파트너 막 단백질의 농도와 표적 막 단백질과 상기 표적 막 단백질에 대한 결합 파트너 막 단백질 간의 복합체의 농도 사이의 비율로서 정의된다.In general, the dissociation constant is defined as the ratio between the concentration of the target membrane protein, the binding partner membrane protein and the concentration of the complex between the target membrane protein and the binding partner membrane protein for the target membrane protein as shown in [Equation 3].
캡처 프로브 처리 전과 비교하여 처리 후에서 증가된 결합 파트너 막 단백질의 부분모집단 비율을 나타내는 ρR은 캡처 프로브 전과 후의 확산계수 분포도에서 부분모집단의 비율을 직접적으로 비교함으로써 구할 수 있다. 보다 구체적으로 단일 분자 수준에서 측정된 확산계수들에 대하여 표준화한 분포도를 그리고, 각 부분모집단에 대하여 하위 면적을 계산함으로써 전체 모집단 대비 부분모집단의 비율을 계산할 수 있다. 캡처 프로브 전과 비교하였을 때, 처리 후의 확산계수 분포도에서 낮은 확산계수를 갖는 부분모집단의 비율이 증가한 정도를 계산함으로써 ρR의 수치값을 구할 수 있다. 캡처 프로브의 처리 전후의 두 개의 확산계수의 분포를 직접적으로 비교하기 위해서는 캡처 프로브 처리 전과 후의 확산계수 분포도가 갖는 전체 단일분자수가 동일해야 하기 때문에 먼저 각 확산계수 분포도의 하위 면적이 1이 되도록 각 그래프를 표준화한다. 확산계수 분포도는 상대적으로 높은 확산계수를 갖는 피크들과 확산 운동이 거의 없는 것으로 간주할 수 있는 낮은 확산계수를 갖는 갖는 피크로 분류되며 각 피크들에 대해 가우시안 그래프(Gaussian graph)로 피팅(fitting)을 수행하면 전체 면적 1 중에서 각 그래프가 갖는 비율을 계산할 수 있다. 그 중 낮은 확산계수를 갖는 피크에 대한 비율을 이용하여 캡처 프로브 처리 전후에 상호작용한 막 단백질의 증가량을 확인할 수 있다. 예를 들어 캡처 프로브의 처리 전에 확산계수 분포도를 그려서 표준화한 후 피팅을 수행했을 때 가우시간 그래프에서 가장 낮은 확산계수를 갖는 피크의 면적이 각각 0.2로 나타났고, 캡처 프로브의 처리 후에 0.5로 변화되었을 때 ρ값은 0.5-0.2=0.3으로 계산한다.Ρ R, which represents the subpopulation ratio of the binding partner membrane protein increased after treatment as compared to before the capture probe treatment, can be obtained by directly comparing the ratio of subpopulations in the diffusion coefficient distribution before and after the capture probe. More specifically, it is possible to calculate the ratio of subpopulations to the entire population by drawing a standardized distribution of the diffusion coefficients measured at the single molecule level and calculating the subarea for each subpopulation. Compared with before the capture probe, the numerical value of ρ R can be obtained by calculating the degree to which the proportion of the subpopulations having the low diffusion coefficient increases in the distribution coefficient distribution after the treatment. In order to directly compare the distributions of the two diffusion coefficients before and after the capture probe treatment, the total single-molecules with the diffusion coefficient distribution before and after the capture probe treatment must be the same. Standardize The diffusion coefficient distribution is divided into peaks with relatively high diffusion coefficients and peaks with low diffusion coefficients that can be considered to have little diffusion motion and are fitted to a Gaussian graph for each peak. By calculating the ratio of each graph of the total area 1 can be calculated. The increase in the amount of membrane protein interacted before and after the capture probe treatment can be confirmed by using a ratio of peaks having a low diffusion coefficient among them. For example, when fitting and performing the diffusion coefficient distribution plot before capturing the capture probe, the area of the peak having the lowest diffusion coefficient in the Gaussian graph was 0.2, respectively, and changed to 0.5 after the capture probe was processed. Ρ value is calculated as 0.5-0.2 = 0.3.
[수학식 3]의 표적 막 단백질, 결합 파트너 막 단백질, 표적 막 단백질과 상기 표적 막 단백질에 대한 결합 파트너 막 단백질 간의 복합체의 농도는 ρR을 활용하여 [수학식 4]와 같이 다시 표현될 수 있으며, 표적 막 단백질의 농도가 표적 막 단백질과 결합 파트너 단백질 간의 복합체 농도보다 매우 높은 경우 [수학식 5]와 같이 단순화하여 정의할 수 있다.The concentration of the complex between the target membrane protein, the binding partner membrane protein, the target membrane protein, and the binding partner membrane protein for the target membrane protein in [Equation 3] can be reexpressed as shown in [Equation 4] using ρ R. If the concentration of the target membrane protein is much higher than the concentration of the complex between the target membrane protein and the binding partner protein, it can be defined simply as shown in [Equation 5].
본 발명에 따른 막 단백질의 상호작용 분석 방법은 후보약물의 처리에 따른 막 단백질 상호작용의 변화를 확인하는 것을 통해 표적 막 단백질에 효과적으로 작용할 수 있는 후보약물을 스크리닝하는데 활용될 수 있다. The method for analyzing the interaction of membrane proteins according to the present invention can be utilized for screening candidate drugs that can effectively act on the target membrane proteins by confirming changes in membrane protein interactions following treatment of candidate drugs.
막 단백질은 약물 개발의 주요 표적이며, 현재 개발된 모든 약물들 중 50% 이상이 막 단백질을 표적으로 하고 있다(Overington, J. P., How many drug targets are there?, Nature reviews. Drug discovery. 5, 993-996, 2006). 예컨대, 심차단(heart block) 또는 서맥(bradycardia)의 치료제인 이소프레날린(isoprenaline)은 adrenergic receptor를 표적으로 하는 약물이며, 당뇨치료제인 인슐린은 insulin receptor를 표적으로 하는 약물로 공지되어 있다(Peter Imming. et al. Drugs, their targets and the nature and number of drug target. Nature reviews. Drug discovery, 2006). 또한 개발된 많은 항암제들은 막 단백질을 표적으로 하고 있다. 예컨대, ErbB family는 암에서 중요한 역할을 하며, 이를 표적으로 하는 항암제의 개발에 대한 중요성은 이미 공지된 상태이며(Eric K. Rowinsky. THE ERBB FAMILY: Targets for Therapeutic Development Against Cancer and Therapeutic Strategies Using Monoclonal Antibodies and Tyrosine Kinase Inhibitors. Annu. Rev. Med. 55, 433-57, 2004), 일례로 대장암 치료제인 세툭시맙(cetuximab)은 ErbB family를 표적으로 하는 약물이다. 따라서, 막 단백질과 리간드와 결합 등에 대한 연구는 표적 막 단백질에 특이적으로 작용하는 약물 개발에 응용될 수 있으며, 구체적으로 표적 막 단백질에 대한 약물을 스크리닝 하는 방법으로 사용될 수 있다. Membrane proteins are a major target for drug development, and more than 50% of all currently developed drugs target membrane proteins (Overington, JP, How many drug targets are there ?, Nature reviews.Drug discovery. 5, 993 -996, 2006). For example, isoprene, a drug for the treatment of heart blocks or bradycardia, is a drug that targets adrenergic receptors, and insulin, a diabetes drug, is known as a drug that targets the insulin receptor (Peter Imming. Et al. Drugs, their targets and the nature and number of drug target.Nature reviews.Drug discovery, 2006). Many of the anticancer drugs developed also target membrane proteins. For example, the ErbB family plays an important role in cancer, and the importance of developing anticancer agents that target it is already known (Eric K. Rowinsky. THE ERBB FAMILY: Targets for Therapeutic Development Against Cancer and Therapeutic Strategies Using Monoclonal Antibodies) and Tyrosine Kinase Inhibitors.Annu. Rev. Med. 55, 433-57, 2004), for example, cetuximab, a colorectal cancer drug, is a drug targeting the ErbB family. Therefore, researches on binding to membrane proteins and ligands, etc. can be applied to the development of drugs that specifically act on the target membrane protein, specifically used as a method for screening drugs for the target membrane protein.
따라서, 본 발명은 추가로 Thus, the present invention further
후보약물의 처리에 따른 살아있는 세포에서 표적 막 단백질과 상기 표적 막 단백질에 대한 결합 파트너 막 단백질 간의 상호작용의 변화를 분석하는 것을 포함하는 표적 막 단백질에 대한 후보약물의 스크리닝 방법에 있어서,A method for screening a candidate drug for a target membrane protein comprising analyzing a change in the interaction between the target membrane protein and a binding partner membrane protein for the target membrane protein in living cells following treatment of the candidate drug,
분석 대상 세포를 기재 상에 위치시키고,Place the cell of interest on the substrate,
상기 분석 대상 세포에 상기 표적 막 단백질에 영향을 미칠 것으로 예상되는 후보약물을 처리하고,Treating the cells of interest with candidate drugs expected to affect the target membrane protein,
기재와 상기 표적 막 단백질 양자에 대한 결합능을 갖는 캡처 프로브를 처리하여 상기 표적 막 단백질을 기재 상에 고정시키고, Fix the target membrane protein on the substrate by treating a capture probe having binding capacity to both the substrate and the target membrane protein,
상기 캡처 프로브의 처리 전과 후의 상기 결합 파트너 막 단백질의 확산계수 분포를 얻고,상기 결합 파트너 막 단백질의 확산계수 분포의 변화를 후보약물 무처리군에서의 캡처 프로브 처리 전과 후의 결합 파트너 막 단백질의 확산계수 분포의 변화와 비교하여 상기 표적 막 단백질과의 상호작용에 대해 영향을 미치는 후보약물을 선별하는 것을 포함하는The diffusion coefficient distribution of the binding partner membrane protein before and after the capture probe is obtained, and the diffusion coefficient distribution of the binding partner membrane protein is changed by the diffusion coefficient of the binding partner membrane protein before and after the capture probe treatment in the candidate drug-free group. Selecting candidate drugs that affect the interaction with the target membrane protein in comparison to the change in distribution.
표적 막 단백질에 대한 후보약물의 스크리닝 방법을 제공한다.Provided are methods for screening candidate drugs for target membrane proteins.
본 발명은 또한 The invention also
후보약물의 처리에 따른 살아있는 세포에서 표적 막 단백질과 상기 표적 막 단백질에 대한 결합 파트너 막 단백질 간의 상호작용의 변화를 분석하는 것을 포함하는 표적 막 단백질에 대한 후보약물의 스크리닝 방법에 있어서,A method for screening a candidate drug for a target membrane protein comprising analyzing a change in the interaction between the target membrane protein and a binding partner membrane protein for the target membrane protein in living cells following treatment of the candidate drug,
분석 대상 세포를 기재 상에 위치시키고,Place the cell of interest on the substrate,
상기 분석 대상 세포에 상기 표적 막 단백질에 영향을 미칠 것으로 예상되는 후보약물을 처리하고,Treating the cells of interest with candidate drugs expected to affect the target membrane protein,
기재와 상기 표적 막 단백질 양자에 대한 결합능을 갖는 캡처 프로브를 처리하여 상기 표적 막 단백질을 기재 상에 고정시키고, Fix the target membrane protein on the substrate by treating a capture probe having binding capacity to both the substrate and the target membrane protein,
상기 캡처 프로브의 처리 전과 후의 상기 결합 파트너 막 단백질의 표적 막 단백질에 대한 해리상수를 얻고,Dissociation constant for the target membrane protein of the binding partner membrane protein before and after treatment of the capture probe,
후보약물 처리에 따른 결합 파트너 막 단백질의 해리상수의 변화를 후보약물 무처리군에서의 캡처 프로브 처리 전과 후의 결합 파트너 막 단백질 해리상수의 변화와 비교하여 상기 표적 막 단백질과의 상호작용에 대해 영향을 미치는 후보약물을 선별하는 것을 포함하는The change in the dissociation constant of the binding partner membrane protein following the candidate drug treatment was compared with the change in the binding partner membrane protein dissociation constant before and after the capture probe treatment in the candidate drug-free group to influence the interaction with the target membrane protein. Affecting the selection of candidate drugs
표적 막 단백질에 대한 후보약물의 스크리닝 방법을 제공한다.Provided are methods for screening candidate drugs for target membrane proteins.
표적 막 단백질에 영향을 미치는 후보약물로는 화합물, 핵산, 당, 탄수화물, 지질, 펩티드 및 단백질로 이루어진 군으로부터 1종 이상을 사용할 수 있다. 상기 단백질은 예를 들어 항체일 수 있으며, 여기에서 항체는 전체 또는 일부, 이를테면 팹(Fab) 부분 또는 힌지 (hinge) 부위만을 선택적으로 잘라낸 부분 (half-immunoglobulin fragment) 모두를 포함하는 개념이다. 한 구체예에서, 상기 후보물질은 핵산일 수 있으며, 여기에서 핵산은 2-200개 염기를 포함하는 올리고 뉴클레오티드 혹은 c-DNA 모두를 포함하는 개념이다. 상기 후보물질은 표적 막 단백질과 결합 파트너 막 단백질 간의 상호작용을 분석하기 위해 적절한 양으로 상기 세포에 처리될 수 있으며, 처리는 당해 분야에 공지된 방법에 따라 수행될 수 있다. 예컨대, 후보물질 0.01 내지 1000 ug/ml를 현탁한 세포배양액을 기재 상에 위치시킨 분석 대상 세포에 직접 처리할 수 있다. Candidate drugs affecting the target membrane protein may be used one or more from the group consisting of compounds, nucleic acids, sugars, carbohydrates, lipids, peptides and proteins. The protein may be, for example, an antibody, wherein the antibody is a concept comprising all or part of all, such as a half-immunoglobulin fragment, which selectively cuts only a Fab portion or a hinge portion. In one embodiment, the candidate may be a nucleic acid, wherein the nucleic acid is a concept comprising both oligonucleotides or c-DNAs comprising 2-200 bases. The candidate may be treated in the cells in an appropriate amount to analyze the interaction between the target membrane protein and the binding partner membrane protein, and the treatment may be performed according to methods known in the art. For example, a cell culture solution containing 0.01 to 1000 ug / ml of the candidate material can be directly treated to the cells of analysis placed on the substrate.
후보약물의 처리에 의해 결합 파트너 막 단백질의 확산계수 분포의 변화가 있거나 결합 파트너 막 단백질의 표적 막 단백질에 대한 해리상수의 변화가 있는 경우, 후보약물은 표적 막 단백질과 결합 파트너 막 단백질 사이의 상호작용에 영향을 미치는 주요한 인자로서 간주할 수 있다. 후보약물의 처리에 의한 결합 파트너 막 단백질의 확산계수 분포의 변화나 해리상수의 변화가 유의수준(significance level)인 경우, 예를 들어, 그러한 변화가 2% 이상, 3% 이상, 5% 이상, 10% 이상, 20% 이상인 경우, 상기 후보물질을 표적 막 단백질과 결합 파트너 막 단백질 사이의 상호작용에 영향을 미치는 약물로 판단할 수 있다. If there is a change in the distribution coefficient distribution of the binding partner membrane protein or change in the dissociation constant of the binding partner membrane protein to the target membrane protein by treatment of the candidate drug, the candidate drug is the interaction between the target membrane protein and the binding partner membrane protein. It can be considered as the main factor affecting the action. If the change in the distribution coefficient distribution or the dissociation constant of the binding partner membrane protein by the treatment of the candidate drug is at the significance level, for example, such a change is at least 2%, at least 3%, at least 5%, In the case of 10% or more and 20% or more, the candidate may be determined as a drug that affects the interaction between the target membrane protein and the binding partner membrane protein.
하기 실시예에서는, 본 발명의 일 실시예로서 캡처 프로브로서 표적 막 단백질에 특이적인 항체를 사용하여 특정 표적 막 단백질만을 기재 상에 고정화하고 다른 막 단백질들은 영향을 받지 않고 움직일 수 있도록 하였다. 자유롭게 움직이는 막 단백질 중에서 표적 막 단백질과 상호작용하는 막 단백질, 즉 결합 파트너 막 단백질은 고정되어 있는 표적 막 단백질과 상호작용이 발생하는 동안 자유롭게 움직이던 막 단백질이 일시적으로 함께 멈추게 되고 이를 통해 복잡한 경로 데이터 중에서 확산계수를 기반으로 일시적인 상호작용에 의한 경로 데이터만을 특이적으로 구별하여 해석할 수 있게 된다. 확산계수 분포도로부터 일시적인 상호작용에 의해 멈추는 단분자 경로들의 숫자를 계산함으로써 전체 분자에 대한 상호작용 복합체를 형성하는 분자들의 비율을 측정할 수 있고 이를 통해 두 단백질 간의 상호작용에 대한 해리상수를 결정할 수 있다. 본 발명자들은 본 발명에 따른 막 단백질의 상호작용 분석 방법을 “공동 면역 고정법(Co-ImmunoImmobilization, Co-II)” 이라고 명명하였다. 이 방법을 이용하면 살아있는 단일 세포의 세포막 위에서 발생하는 리간드 의존적/독립적인 막 단백질 간의 상호작용을 정량적으로 분석할 수 있으며, 이를 통해 단백질의 구조와 세포막이 형성하는 미세환경 등에 의해 복합적으로 조절되는 막 단백질 간의 상호작용에 대해 밝혀낼 수 있다.In the following examples, as an embodiment of the present invention, an antibody specific for a target membrane protein as a capture probe was used to immobilize only a specific target membrane protein on a substrate and allow other membrane proteins to move unaffected. Among freely moving membrane proteins, the membrane protein that interacts with the target membrane protein, that is, the binding partner membrane protein, temporarily stops freely moving membrane proteins while interacting with the immobilized target membrane protein. Based on the diffusion coefficient, only the path data by the temporary interaction can be distinguished and interpreted. By calculating the number of single-molecule pathways stopped by transient interactions from the diffusion coefficient distribution chart, we can measure the ratio of molecules forming the interaction complex to the whole molecule, and determine the dissociation constant for the interaction between the two proteins. have. The inventors named the method for analyzing the interaction of membrane proteins according to the present invention as "Co-ImmunoImmobilization (Co-II)". This method can be used to quantitatively analyze the interaction between ligand-dependent / independent membrane proteins that occur on the cell membrane of living single cells, which is a complex membrane controlled by the protein structure and the microenvironment formed by the cell membrane. Interactions between proteins can be revealed.
본 발명에 따르면, 살아있는 세포에서 표적 막 단백질과 상기 표적 막 단백질에 대한 결합 파트너 막 단백질 간의 상호작용을 분석하는 방법에 있어서, 표적 막 단백질에 대한 캡처 프로브를 분석 대상 세포에 처리하여 상기 표적 막 단백질을 기재 상에 고정시키고 상기 표적 막 단백질에 대한 결합 파트너 막 단백질의 확산계수 분포의 변화를 측정함으로써 상호작용에 대한 해리상수를 계산할 수 있으며 따라서 표적 막 단백질과 상기 표적 막 단백질에 대한 결합 파트너 막 단백질 간의 상호작용을 정량적으로 측정하는 것이 가능하게 된다.According to the present invention, in a method for analyzing an interaction between a target membrane protein and a binding partner membrane protein for the target membrane protein in living cells, the target membrane protein is treated by treating a target probe protein with a capture probe for the target membrane protein. Can be calculated on the substrate and the dissociation constant for the interaction can be calculated by measuring the change in the distribution coefficient distribution of the binding partner membrane protein for the target membrane protein and thus the target membrane protein and the binding partner membrane protein for the target membrane protein. It is possible to measure the interaction between them quantitatively.
도 1은 확산계수를 기반으로 살아있는 단일 세포에서 막 단백질 간의 일시적인 상호작용을 정량적으로 조사하는 방법에 대한 모식도를 나타낸다. (a) 항체를 처리하기 전에 형광으로 표지된 먹이(Prey) 단백질은 상호작용 상태에 상관없이 다양한 확산운동을 하고 있기 때문에 특정한 상호작용 복합체를 확산만으로 구분해내는 것은 불가능하다. 미끼(Bait) 단백질에 특이적으로 결합하는 항체를 처리하여 커버슬립 표면에 미끼 단백질을 고정하게 되면 먹이 단백질 중 미끼 단백질과 상호작용하고 있는 부분모집단만이 미끼 단백질과 함께 고정화된다. (b) 형광으로 표지된 먹이 단백질의 단분자 경로를 추적할 때, 미끼 특이적인 항체의 처리에 의해 낮은 확산계수를 갖는 경로들이 증가하게 되는데 이 경로들의 수는 곧 고정화된 미끼 단백질과 상호작용하여 형성한 상호작용 복합체의 양을 의미하게 된다. 이 부분모집단의 비율은 먹이 단백질의 확산계수 분포도에서 증가된 낮은 확산계수를 갖는 부분모집단 비율(ρR)로서 계산된다. (c) 결합 파트너 막 단백질 R과 표적 막 단백질 I 사이에서 일시적인 상호작용을 통해 형성되는 복합체 RI의 해리상수(KD)는 단백질 I를 커버슬립 위에 고정한 후에 증가한 고정화된 부분모집단의 비율 ρR을 측정함으로써 계산될 수 있다. 1 shows a schematic diagram of a method for quantitatively examining the transient interaction between membrane proteins in living single cells based on the diffusion coefficient. (a) It is impossible to distinguish specific interaction complexes by diffusion only because the fluorescently labeled prey proteins undergo various diffusion movements regardless of the interaction state. If the bait protein is fixed to the cover slip surface by treating an antibody that specifically binds to the bait protein, only a subset of the food proteins interacting with the bait protein is immobilized together with the bait protein. (b) When tracking single molecule pathways of fluorescently labeled food proteins, pathways with low diffusion coefficients are increased by treatment of bait-specific antibodies, which in turn interact with immobilized bait proteins The amount of interaction complexes formed. The proportion of this subpopulation is calculated as the subpopulation ratio (pR) with a low diffusion coefficient increased in the distribution coefficient distribution of the feed protein. (c) The dissociation constant (K D ) of the complex RI formed through the transient interaction between the binding partner membrane protein R and the target membrane protein I measures the ratio ρR of the increased immobilized subpopulation after immobilizing protein I on the coverslip. Can be calculated.
도 2는 2 is 커버슬립Coverslip 표면으로부터 고정된 항체의 높이에 따른  Depending on the height of the immobilized antibody from the surface EGFREGFR 고정화 정도의 의존성을 나타내는 모식도와 실험 결과를 보여준다. The schematic and the experimental results showing the dependence of the degree of immobilization are shown.
(a-c) 커버슬립 표면에 고정시킨 항체의 숫자(항체 1개 (a), 항체 2개 복합체 (b), 항체 3개 복합체 (c))에 따라 각기 다른 높이를 갖는 EGFR 단백질 고정화 시스템의 도해. (d) EGFR이 발현된 COS7 세포에 EGFR 특이적인 항체를 처리했을 때 EGFR의 고정화된 부분모집단의 비율.(a-c) Illustration of EGFR protein immobilization systems with different heights depending on the number of antibodies immobilized on the coverslip surface (1 antibody (a), 2 antibody complexes (b), 3 antibody complexes (c)). (d) Proportion of immobilized subpopulations of EGFR when treated with EGFR specific antibodies in COS7 cells expressing EGFR.
도 3은 고정화의 막 단백질3 Membrane protein of immobilization 의of 발현량에 대한 독립성을 보여주는 실험 결과를 나타낸다. Experimental results showing independence of the amount of expression are shown.
(a) mEos3.2-EGFR이 발현된 COS7 세포는 mEos3.2의 형광 세기기준의 발현 정도에 따라 low (#1), middle (#2), and high (#3) 로 나누어 분류되었다. (b) 웨스턴 블롯을 이용하여 분류된 세 집단의 EGFR 발현 정도를 확인하였다. 1번 낮은 발현 집단은 내재적 EGFR의 발현과 유사한 정도를 보였고, 2번 중간 발현 및 3번 높은 발현 집단은 내재적 EGFR의 발현보다 훨씬 더 높은 발현 정도를 보였다. (c) mEos3.2의 형광 세기로 계산된 mEos3.2 항체 처리 전(빈 원) 후(채워진 원)의 mEos3.2-EGFR의 고정화 정도는 발현 정도에 따른 세 집단 (검정색-낮은 발현; 파란색-중간 발현, 빨간색-높은 발현) 모두에서 일정한 수준으로 나오는 것을 확인할 수 있었다.(A) COS7 cells expressing mEos3.2-EGFR were divided into low (# 1), middle (# 2), and high (# 3) according to the expression level of mEos3.2 fluorescence intensity. (b) Western blot was used to determine the extent of EGFR expression in three groups. The first low expression population showed similar levels of expression of the endogenous EGFR, and the second and third high expression populations showed much higher expression levels than the expression of the endogenous EGFR. (c) The degree of immobilization of mEos3.2-EGFR before (empty circle) (filled circle) before mEos3.2 antibody treatment calculated as the fluorescence intensity of mEos3.2 was determined in three groups (black-low expression; blue; -Intermediate expression, red-high expression) were found to come out at a constant level.
도 4는 다양한 항체 사용시 고정화 정도의 효율성을 확인한 결과를 나타낸다.Figure 4 shows the results confirming the efficiency of the degree of immobilization when using various antibodies.
(a-c) COS7세포에서 EGFR의 서로 다른 결합부위에 특이적으로 결합하는 mAb 199.12 (a), mAb R-1 (b), 및 mAb 528 (c) 등의 항체들을 처리하였을 때의 EGFR의 고정화 비율. (d) SNAP-EGFR이 발현된 COS7세포에 SNAP 항체 처리 전후의 고정화된 부분모집단의 비율. (e,f) N말단(e)과 C말단(f)에 mEos3.2 형광 단백질이 연결된 EGFR이 발현된 COS7 세포에 mEos3.2 단백질 특이적 항체를 처리하였을 때의 고정화된 부분모집단의 비율. 각 검정색 점은 단세포에서 측정된 고정화 비율을 의미하고, 빨간색 선은 평균값을 의미한다.(ac) Immobilization rate of EGFR when treated with antibodies such as mAb 199.12 (a), mAb R-1 (b), and mAb 528 (c) that specifically bind to different binding sites of EGFR in COS7 cells . (d) Proportion of immobilized subpopulations before and after SNAP antibody treatment in COS7 cells expressing SNAP-EGFR. (e, f) Proportion of immobilized subpopulations when mEos3.2 protein specific antibodies were treated to COS7 cells expressing EGFR linked to mEos3.2 fluorescent proteins at N-terminus (e) and C-terminus (f). Each black dot represents the rate of immobilization measured in single cells, and the red line represents the mean value.
도 5는 다양한 세포주에서 고정화 정도의 효율성을 확인한 결과를 나타낸다.5 shows the results of confirming the efficiency of the degree of immobilization in various cell lines.
EGFR-mEos3.2가 발현된 각각의 COS7세포, HEK293세포, HeLa세포, CHO-K1세포에서 EGFR 특이적 항체의 처리 전후에 고정화된 EGFR의 비율을 측정하였다. 모든 세포주에서 고정화된 EGFR의 비율은 비슷함을 알 수 있다.The ratio of immobilized EGFR in each COS7 cell, HEK293 cell, HeLa cell, CHO-K1 cell expressing EGFR-mEos3.2 before and after treatment with the EGFR specific antibody was measured. It can be seen that the ratio of immobilized EGFR in all cell lines is similar.
도 6은 살아있는 세포의 세포막에서 분자 특이적인 고정화를 실험한 결과를 나타낸다. (a,b) EGFR 특이적인 항체 처리 전후의 COS7세포에서 동시에 발현된 SNAP-EGFR와 β2-AR-mEos3.2 도해 및 경로 지도. 5,000개의 경로가 한 개의 경로 지도에 그려져있다. Scale bar, 5μm. (c) EGFR 특이적인 항체 처리 전(검은 선)과 후(붉은 선)의 SNAP-EGFR(위)와 β2-AR-mEos3.2(아래)의 확산계수 분포도. 고정화되어 나타나는 부분모집단을 구분하는 확산계수 기준은 파란 세로선으로 표시되어 있다. (d) IgG와 EGFR 특이적인 항체 처리 전후의 SNAP-EGFR(검은 바)와 β2-AR-mEos3.2(붉은 바)의 고정화된 부분모집단의 비율(%). (e) EGFR 특이적인 항체 처리 전후의 PMT, EGFR, ErbB2, ErbB3, InsR, 및 β2-AR의 고정화된 부분모집단의 비율(%). 단일 점은 하나의 세포로부터 얻은 데이터이며 붉은 가로 선은 평균값을 의미한다. n.s. 는 통계적으로 유의미하지 않음을 뜻한다. **P < 0.0001 and *P<0.01 (Student's t-test). Figure 6 shows the results of experiments with molecular specific immobilization in the cell membrane of living cells. (a, b) Schematic and pathway map of SNAP-EGFR and β2-AR-mEos3.2 coexpressed in COS7 cells before and after EGFR specific antibody treatment. 5,000 routes are drawn on one route map. Scale bar, 5 μm. (c) Diffusion coefficient distribution of SNAP-EGFR (top) and β2-AR-mEos3.2 (bottom) before and after EGFR specific antibody treatment (black line). The diffusion coefficient criterion for distinguishing a subpopulation that appears to be fixed is indicated by a blue vertical line. (d) Percentage of immobilized subpopulations of SNAP-EGFR (black bars) and β2-AR-mEos3.2 (red bars) before and after IgG and EGFR specific antibody treatment. (e) Percentage of immobilized subpopulations of PMT, EGFR, ErbB2, ErbB3, InsR, and β2-AR before and after EGFR specific antibody treatment. Single points are data from one cell and the red horizontal line represents the mean value. ns means it is not statistically significant. ** P <0.0001 and * P <0.01 (Student's t -test).
도 7은 EGFR 전-동종 이합체 형성에서 일시적인 상호작용의 해리상수를 측정한 결과를 나타낸다. (a) EGFR 전-동종 이합체에 대한 해리상수의 측정을 위한 시스템 도해. EGFR-mEos3.2는 움직이는 상태에 있다가 고정화된 SNAP-EGFR과 상호작용을 할 때에만 움직이지 않는 상태로 있게 된다. (b) 동일 COS7 세포에서 SNAP tag 특이적인 항체 처리 전과 후의 EGFR-mEos3.2(왼쪽)과 SNAP-EGFR(오른쪽)의 경로 지도. 100개의 경로가 각 경로 지도에 표시되었다. Scale bar, 2μm. (c) SNAP tag 특이적인 항체 처리 전(검은 선)과 후(붉은 선)의 SNAP-EGFR과 EGFR-mEos3.2의 확산계수 분포. (d, e) SNAP tag 특이적인 항체 처리 전과 후의 SNAP-EGFR(d)과 EGFR-mEos3.2(e)의 고정화된 부분모집단의 비율. ρI는 항체 처리에 의해 증가한 EGFR-mEos3.2의 고정화된 부분모집단의 비율을 의미한다. (f) SNAP-EGFR에 대한 전체 및 단분자 발현 수준에 대한 형광 이미지. Scale bar, 5μm and 2μm. 단분자는 이미징 중에 형광 세기 그래프에서 단일 광탈색 단계를 보이는 것으로 구분된다. (g) COS7 세포에서 EGFR 전-동종 이합체의 해리상수는 SNAP-EGFR의 발현 수준에 독립적이며 그 평균값은 약 2727±126molecules/μm2이다. (h) SNAP-EGFR의 발현량에 따른 EGFR-mEos3.2의 고정화된 부분모집단의 비율 및 EGFR-mEos3.2의 고정화된 부분모집단의 비율에 따른 SNAP-EGFR의 결합/비결합 비율을 나타낸 그래프이다. (i) 다양한 세포주에서 측정된 해리상수. 7 shows the results of measuring the dissociation constants of transient interactions in EGFR pre- homodimer formation . (a) System diagram for the measurement of dissociation constants for EGFR pre-homogenous dimers. EGFR-mEos3.2 remains in motion only when it interacts with immobilized SNAP-EGFR. (b) Route map of EGFR-mEos3.2 (left) and SNAP-EGFR (right) before and after SNAP tag specific antibody treatment in the same COS7 cells. 100 routes were marked on each route map. Scale bar, 2 μm. (c) Distribution of diffusion coefficients for SNAP-EGFR and EGFR-mEos3.2 before (black line) and after (red line) SNAP tag specific antibody treatment. (d, e) Ratio of immobilized subpopulations of SNAP-EGFR (d) and EGFR-mEos3.2 (e) before and after SNAP tag specific antibody treatment. ρ I means the proportion of immobilized subpopulations of EGFR-mEos3.2 increased by antibody treatment. (f) Fluorescence images of total and monomolecular expression levels for SNAP-EGFR. Scale bar, 5 μm and 2 μm. Monomolecules are distinguished by showing a single photobleaching step in the fluorescence intensity graph during imaging. (g) The dissociation constant of EGFR pre-homogenous dimers in COS7 cells is independent of the expression level of SNAP-EGFR and its mean value is about 2727 ± 126 molecules / μm 2 . (h) Graph showing the ratio of binding / non-binding ratio of SNAP-EGFR to the ratio of immobilized subpopulations of EGFR-mEos3.2 and the ratio of immobilized subpopulations of EGFR-mEos3.2 according to the amount of expression of SNAP-EGFR. to be. (i) Dissociation constants measured in various cell lines.
도 8은 고정된 SNAP-8 shows a fixed SNAP- EGFR과With EGFR EGFREGFR -- mEos3mEos3 .2 경로가 공존하고 있는 양상을 보여주는 사진이다..2 Photograph showing how paths coexist.
빨간색 선은 EGFR-mEos3.2 단분자 중 하나의 경로를 의미하고, 흰색 점은 SNAP tag 특이적 항체에 의해 고정화된 SNAP-EGFR을 의미한다. 고정화된 SNAP-EGFR의 위치와 일시적으로 고정화되는 EGFR-mEos3.2의 경로 일부가 완전히 동일한 위치에서 공존함을 알 수 있다.The red line represents the pathway of one of the EGFR-mEos3.2 monomolecules, and the white dot represents SNAP-EGFR immobilized by SNAP tag specific antibodies. It can be seen that the position of the immobilized SNAP-EGFR and part of the pathway of the temporarily immobilized EGFR-mEos3.2 coexist at exactly the same position.
도 9는 고정화된 막 단백질의 총 농도를 정량한 실험 결과를 나타낸다.9 shows experimental results of quantifying the total concentration of immobilized membrane protein.
(a) SNAP-CF660R 과 Cetuximab-Alexa fluor 647을 이용해 얻은 총 형광 신호와 단분자 형광 신호 이미지. HeLa세포에 SNAP-EGFR을 발현시키고 CF660R을 결합시켜 전체 형광 신호 이미지를 얻고 충분한 광탈색 후 단분자 형광 신호 이미지를 얻었다. CF660R의 완전한 광탈색 이후, Alexa fluor 647 염료가 결합되어 있는 EGFR 특이적 항체인 Cetuximab을 처리하여 세포 표면에 발현되어 있는 모든 EGFR을 레이블링하여 유사한 방식으로 전체 형광 신호 이미지와 단분자 형광 신호 이미지를 얻었다. (b) 원 이미지로부터 정량화된 단백질의 농도 비율. ImageJ 프로그램을 이용해 원 이미지로부터 형광 신호를 정량화 하였고, 단백질의 농도는 총 형광신호를 단분자의 형광신호로 나누어 줌으로써 계산하였다. 동일한 세포의 EGFR 대해서 CF660R-SNAP를 이용해 구한 결과와 Cetuximab Alexa flour 647을 이용해 구한 단백질의 농도는 0.91±0.13의 비율을 나타내었다.(a) Total fluorescence and monomolecular fluorescence signal images obtained using SNAP-CF660R and Cetuximab-Alexa fluor 647. SNAP-EGFR was expressed in HeLa cells and CF660R was combined to obtain a whole fluorescence signal image, and a single molecule fluorescence signal image was obtained after sufficient photobleaching. After complete photobleaching of CF660R, Cetuximab, an EGFR specific antibody bound to Alexa fluor 647 dye, was treated to label all EGFR expressed on the cell surface to obtain a full fluorescence signal image and a single molecule fluorescence signal image in a similar manner. . (b) Concentration percentage of protein quantified from the original image. The fluorescence signal was quantified from the original image using the ImageJ program, and the protein concentration was calculated by dividing the total fluorescence signal by the fluorescence signal of a single molecule. The protein concentration obtained from CF660R-SNAP and Cetuximab Alexa flour 647 for EGFR of the same cell was 0.91 ± 0.13.
도 10은 막 단백질의 발현 정도에 따른 고정화된 부분모집단의 비율의 의존성을 확인한 결과를 보여준다.10 shows the results of confirming the dependence of the ratio of the immobilized subpopulation according to the expression level of the membrane protein.
COS7세포 내 발현된 SNAP-EGFR에 SNAP tag 특이적 항체를 처리한 후 고정화된 부분모집단의 비율은 발현 수준에 따라 양의 상관관계를 나타낸다. 데이터 상 하나의 점은 하나의 세포에서 나온 정보를 나타낸다.The ratio of immobilized subpopulations after treatment of SNAP tag specific antibodies to SNAP-EGFR expressed in COS7 cells shows a positive correlation with expression levels. One dot in the data represents information from one cell.
도 11은 11 is EGFREGFR 고정화 후에도 영향을 받지 않는  Unaffected even after immobilization EGFR의EGFR 신호전달을 확인한 결과를 보여준다. Shows the result of checking signal transmission.
COS7 세포에 발현된 mEos3.2-EGFR은 mEos3.2 단백질 특이적 항체를 이용해 커버슬립에 고정되었다. 유사한 수준의 mEos3.2-EGFR 발현 수준을 갖는 세포에 대해서 EGF 유무에 따른 EGFR의 인산화 정도를 EGFR의 Y1068 아미노산의 인산화기 항체와 Alexa 647이 결합된 2차 항체를 사용해 측정하였다. ImageJ 프로그램을 사용해 원 이미지로부터 형광 신호를 정량화하였다. EGFR이 고정화 되어 있음에도 불구하고 NT와 비교시 EGF 처리된 환경에서 EGFR의 인산화 정도가 유의미하게 증가하였다.MEos3.2-EGFR expressed in COS7 cells was immobilized on the coverslip using mEos3.2 protein specific antibodies. For cells with similar levels of mEos3.2-EGFR expression, the degree of phosphorylation of EGFR with or without EGF was measured using a phosphorylated antibody of Y1068 amino acid of EGFR and a secondary antibody combined with Alexa 647. Fluorescence signals were quantified from the original image using the ImageJ program. Despite the immobilization of EGFR, the phosphorylation of EGFR was significantly increased in the EGF-treated environment compared to NT.
도 12는 EGFR 동종이합체의 해리상수에 대한 티로신 인산화 저해제의 직접적인 결합의 효과를 확인한 결과이다. (a) 굶긴 COS7세포에서 mock, Erlotinib, Lapatinib의 처리 후에 EGF 유무에 따른 EGFR 동종이합체의 해리상수. (b) EGFR의 이합체 형성에 있어서 티로신 인산화 저해제(TKI)의 효과에 대한 도해. TKI는 EGFR에 결합하여 세포 안쪽의 구조에 영향을 줌으로써 이 부위를 통한 EGFR의 결합을 촉진하고 이를 통해 EGF가 없는 상황에서도 EGFR 이합체 형성에 유리한 환경을 형성한다. 12 is EGFR Homodimeric The effect of direct binding of tyrosine phosphorylation inhibitor on dissociation constant was confirmed. (a) Dissociation constant of EGFR homodimer with or without EGF after treatment with mock, Erlotinib, and Lapatinib in starved COS7 cells. (b) Illustration of the effect of tyrosine phosphorylation inhibitor (TKI) on dimer formation of EGFR. TKI binds to EGFR and affects the structure inside the cell, facilitating the binding of EGFR through this site, thereby creating a favorable environment for EGFR dimer formation even in the absence of EGF.
도 13은 EGFR 동종 이합체의 해리상수에 대한 세포막의 환경변화의 효과를 확인한 결과이다. (a) 굶긴 COS7세포에서 Latrunculin B의 처리 후에 EGF 유무에 따른 EGFR 동종 이합체의 해리상수. (b) 액틴 구조의 붕괴에 따른 EGF에 의해 유도된 EGFR 이합체 형성에의 효과에 대한 도해. 액틴 골격은 EGFR 이합체를 안정화하는 기능을 수행하기 때문에 Latrunculin B의 처리에 의해 액틴이 부서지게 되면 EGFR 이합체의 안정성이 감소하여 그 비율이 감소하게 된다. (c) 굶긴 COS7세포에서 Nystatin의 처리 후에 EGF 유무에 따른 EGFR 동종 이합체의 해리상수. (d) 콜레스테롤 제거에 따른 EGFR 전-이합체 형성에의 효과에 대한 도해. EGFR의 세포밖 부위는 세포막의 콜레스테롤과 상호작용하여 접힌 형태를 보이는 경향성을 가진다. Nystatin에 의해 콜레스테롤이 제거되면 EGFR 단량체가 펼쳐진 형태로 자유롭게 되어 리간드의 결합 없이도 EGFR 이합체 형성을 촉진하게 된다. 13 is the same kind of EGFR dimers The result of confirming the effect of environmental changes of the cell membrane on dissociation constant . (a) Dissociation constant of EGFR homodimer with and without EGF after treatment with Latrunculin B in starved COS7 cells. (b) Illustration of the effect on the formation of EGFR dimers induced by EGF following disruption of actin structure. Actin backbone acts to stabilize the EGFR dimer, so when actin is broken by the treatment of Latrunculin B, the stability of the EGFR dimer decreases and the ratio decreases. (c) Dissociation constant of EGFR homodimer with or without EGF after Nystatin treatment in starved COS7 cells. (d) Illustration of the effect on EGFR pre-dimer formation following cholesterol removal. Extracellular sites of EGFR tend to interact with cholesterol in the cell membrane to show folded forms. When cholesterol is removed by nystatin, the EGFR monomer is freed into unfolded form to promote EGFR dimer formation without binding of ligand.
이하에서, 본 발명을 실시예를 통해 상세히 설명한다. 하기 실시예는 본 발명을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail through examples. The following examples are merely illustrative of the present invention, and the scope of the present invention is not limited to the following examples.
실험예 1. 공동 면역 고정법의 원리Experimental Example 1. Principle of joint immunofixation
살아있는 단일 세포에서 세포막 단백질들간의 상호작용을 정량적으로 측정하기 위해 공동 면역 고정법은 커버슬립에 코팅되어 있는 특이적인 항체에 의해 고정된 '미끼' 단백질(표적 막 단백질)을 사용한다. 이 상황에서 '먹이' 단백질(상호작용 후보 단백질, 즉 결합 파트너 막 단백질)은 자유롭게 운동하고 있다가 고정된 미끼 단백질과 상호작용하게 되면 일시적으로 함께 멈추게 된다(도 1a). 이러한 일시적인 상호작용의 순간은 광전환이 가능한 형광 단백질이나 유기 형광 염료를 이용하여 초해상도 현미경을 활용한 단일 추적 방법으로 관찰할 수 있다. 이를 통해 살아있는 하나의 세포에서 상호작용에 대한 정량적인 정보를 얻어내기 위해 통계적으로 충분한 수의 경로 데이터를 얻을 수 있다. 미끼 단백질을 고정화하기 위한 항체 처리 전과 후의 조건에서 먹이 단백질의 확산계수 분포를 비교함으로써 일시적인 상호작용에 의해 증가한 고정화된 경로들의 부분모집단의 양을 정량화함으로써 미끼 단백질과 먹이 단백질이 이루는 복합체의 양을 계산할 수 있다(도 1b). 두 단백질의 상호작용에 대한 해리상수를 구하기 위해서는 일반적으로 두 단백질의 해리된 상태에서의 농도와 평형상태에서의 두 단백질의 복합체 농도의 세 가지 변수가 필요하다. 우리는 여기에서 움직이는 먹이 단백질인 R과 고정화된 미끼 단백질인 I 사이에서 일어나는 상호작용을 측정하기 때문에(도 1c) 기술적으로 해리상수는 다음 [수학식 3]과 같이 정의될 수 있다.To quantitatively measure the interaction between membrane proteins in living single cells, co-immuno immobilization uses 'bait' proteins (target membrane proteins) immobilized by specific antibodies coated on coverslips. In this situation, the 'feed' proteins (interaction candidate proteins, ie binding partner membrane proteins) are free to move and temporarily stop together when interacting with the immobilized bait protein (FIG. 1A). These moments of interaction can be observed with a single tracking method using a super resolution microscope using fluorescent proteins or organic fluorescent dyes that can be converted. This provides a statistically sufficient number of pathway data to obtain quantitative information about interactions in a single living cell. Calculate the amount of complex between the bait protein and the feed protein by quantifying the amount of subpopulations of immobilized pathways increased by transient interactions by comparing the distribution of the distribution coefficients of the feed protein under conditions before and after antibody treatment to immobilize the bait protein. May be (FIG. 1B). In order to determine the dissociation constant for the interaction of two proteins, three variables are generally required: the concentration of the two proteins in the dissociated state and the concentration of the complex of the two proteins in equilibrium. Since we measure the interaction between the moving feed protein R and the immobilized bait protein I here (Fig. 1c), the dissociation constant can be technically defined as Equation 3 below.
KD = [R][I] / [RI] … [수학식 3]K D = [R] [I] / [RI]. [Equation 3]
고정화된 미끼 단백질의 양이 복합체의 양보다 월등히 많은 경우, 동역학적인 평형상태에 도달했을 때 미끼 단백질의 절대 농도는 초기 농도와 같다고 가정할 수 있다. 따라서 [수학식 3]에서 [R]/[RI]가 무차원의 값인 미끼 단백질과 결합하지 않은 먹이 단백질과 결합한 먹이 단백질의 비율로 치환됨으로써 먹이 단백질의 절대적인 농도 정보가 필요하지 않게 되고, 이에 따라 2차 이분자 반응 수학식은 유사 1차 반응으로 계산될 수 있게 된다. 미끼 단백질을 고정하기 전과 비교하였을 때, 고정화 후에 증가한 둘 사이의 상호작용에 의해 고정화된 확산계수를 가지는 먹이 단백질의 비율 ρR을 측정함으로써 이 값을 바로 먹이 단백질의 미끼 단백질에 대한 비결합/결합 비율로 치환하여 사용할 수 있다. 따라서 해리상수를 구하기 위한 [수학식 3]은 본 발명에서 제안하는 실험 디자인에서 다음의 [수학식 5]와 같이 변환될 수 있다. (더 자세한 치환 과정은 Methods에서 확인할 수 있다.)If the amount of immobilized bait protein is significantly greater than the amount of complex, it can be assumed that the absolute concentration of the bait protein is equal to the initial concentration when the dynamic equilibrium is reached. Therefore, in [Equation 3], [R] / [RI] is substituted by the ratio of the feed protein that is not bound to the bait protein, which is a dimensionless value, and does not need the absolute concentration information of the feed protein. The second order bimolecular response equation can be calculated as a pseudo first order reaction. This value is determined by determining the ratio ρ R of the feed protein with the diffusion coefficient immobilized by the interaction between the two increased after immobilization, as compared with before the fixation of the bait protein. It can substitute and use in ratio. Therefore, Equation 3 for obtaining the dissociation constant may be converted as shown in Equation 5 in the experimental design proposed by the present invention. (More substitution procedures can be found in the methods.)
KD = (1-ρR)[I]0 / ρR … [수학식 5]K D = (1-ρ R ) [I] 0 / ρ R. [Equation 5]
그러므로 살아있는 단일 세포에서 공동 면역 고정법을 이용하여 두 세포막 단백질 사이의 해리상수를 계산하기 위해서는 [수학식 5]에서 보는 것과 같이 크게 2개의 변수가 요구된다.Therefore, two variables are required to calculate the dissociation constants between two membrane proteins using co-immunoimmobilization in living single cells as shown in [Equation 5].
1) 미끼 단백질의 고정화 전과 비교하여 처리 후의 조건에서 증가된 먹이 단백질의 고정화 비율1) Increased feed protein immobilization rate under conditions after treatment as compared to before bait protein immobilization
2) 고정화된 미끼 단백질의 절대적 농도2) absolute concentration of immobilized bait protein
실험예 2. 살아있는 세포의 세포막에서 막 단백질의 분자 특이적 고정화Experimental Example 2 Molecular Specific Immobilization of Membrane Proteins in the Cell Membrane of Living Cells
미끼 단백질이 항체에 의해 커버슬립에 고정화되는 효율은 공동 면역 고정법의 민감도에 중요한 요소이다. 따라서 상피세포 성장인자 수용체(EGFR)을 사용하여 이 효율을 측정하였다.The efficiency with which the bait protein is immobilized on the coverslip by the antibody is an important factor for the sensitivity of joint immunofixation. Therefore, epithelial growth factor receptor (EGFR) was used to measure this efficiency.
먼저 EGFR 특이적인 항체가 코팅되어 있는 커버슬립을 만들기 위해서 3-머캅토프로필트리메톡시실란(3-mercaptopropyltrimethoxysilane)을 사용하여 티올 반응기가 활성화되어 있는 커버슬립을 준비하였다. 그 다음, 말레이미드(maleimide)가 활성화되어 있는 뉴트라비딘(neutravidin)을 처리하여 반응기 사이의 공유결합을 유도함으로써 뉴트라비딘이 결합된 커버슬립을 만든 후 여기에 마지막으로 비오틴이 결합되어 있는 EGFR 특이적인 항체를 처리하였다. First, to prepare a coverslip coated with an EGFR specific antibody, 3-mercaptopropyltrimethoxysilane was used to prepare a coverslip in which a thiol reactor was activated. Next, neutravidin with maleimide is activated to induce covalent bonds between the reactors to make neutravidin-bound coverslips, and finally to EGFR-specific biotin-bound Antibodies were treated.
COS7 세포에서 C말단에 mEos3.2가 연결된 EGFR(EGFR-mEos3.2)을 발현시킨 다음, 다중 입자 추적 방법을 사용하여 biotin이 결합되어 있는 EGFR 특이적인 항체의 처리로 인해 발생하는 EGFR의 고정화된 부분모집단의 비율을 측정하였다(도 2a). 의도적으로 발현시킨 EGFR뿐 아니라 내재적으로 발현되어 있는 EGFR까지 고정화할 수 있는 충분한 양의 항체를 처리하였음에도 불구하고 고정화된 EGFR의 비율은 ~61% 정도로 충분하지 않았다(도 2d). 일반적으로 50nm 이상에 달하는 세포막과 커버슬립 표면 사이의 거리에 반해 말레이미드, 비오틴, 그리고 항체가 연결되어 이루는 높이는 커버슬립 표면으로부터 약 ~30nm이며 EGFR의 높이는 약 ~15nm에 달한다고 추측되므로 이 정도 높이로는 커버슬립에 밀착되어 있는 세포에서 일부 표면에만 물리적으로 결합할 수 있어 EGFR이 충분히 고정화되지 못했다고 판단되었다. 따라서 우리는 뉴트라비딘과 EGFR 특이적인 항체 사이에 한 개의 추가적인 항체(2차 항체)를 처리하여 커버슬립으로부터의 높이를 높이고자 하였다(도 2b). 2차 항체를 사용함으로써 예상되는 높이는 약 ~45nm가 되었고 그 결과 커버슬립에 잘 부착된 COS7세포에서 >97.3%의 EGFR이 고정화었다(도 2d). 추가적으로 3차 항체를 도입하여 예상 높이를 ~60nm까지 높였을 때에도 EGFR의 고정화 비율에 있어서 유의미한 개선은 관찰할 수 없었다(도 2c,d). 2차 항체까지 활용하여 커버슬립에 코팅한 EGFR 특이적인 항체의 양은 COS7 세포에서 넓은 범위의 발현 수준에서 모든 EGFR을 고정화시키기에 충분하였다(도 3). 또한 mAb 199.12, mAb R-1, mAb 528 등과 같이 EGFR의 서로 다른 부위에 결합하는 다양한 항체를 사용하여 고정화에 사용되는 EGFR 특이적인 항체의 종류를 달리하여도 비슷하게 충분한 수준으로 EGFR이 고정화되는 것을 확인하였다(도 4a-c). 추가적으로 EGFR의 N말단에 SNAP tag이 결합된 EGFR(SNAP-EGFR)과 mEos3.2 단백질이 결합된 EGFR(mEos3.2-EGFR)을 가지고 각각 SNAP tag 특이적인 항체와 mEos3.2 단백질 특이적인 항체에 대한 고정화 효율을 알아보았다. SNAP-EGFR과 mEos3.2-EGFR은 각각 SNAP tag 특이적인 항체와 mEos3.2 단백질 특이적인 항체에 대해서 거의 완전한 고정화 효율을 보였지만, EGFR의 C말단에 mEos3.2가 연결된 EGFR-mEos3.2는 mEos3.2 단백질 특이적 항체에 의해 전혀 영향을 받지 않았다(도 4d-f). 뿐만 아니라, HEK293세포, HeLa세포 및 CHO-K1세포 등의 서로 다른 세포주에서도 90% 이상의 EGFR이 효과적으로 고정화됨을 확인하였다(도 5). 위와 같은 결과를 통해 커버슬립 표면으로부터 세포에 닿기까지 항체의 높이만 충족한다면, 항체의 결합 부위나 세포막의 미세환경의 차이는 고정화의 효율에 크게 영향을 주지 않는다는 것을 알 수 있다.EGFR (EGFR-mEos3.2) linked to the C-terminus at the C-terminus in COS7 cells, and then immobilized EGFR resulting from treatment of EGFR-specific antibodies with biotin bound using a multi-particle tracking method The proportion of subpopulations was measured (FIG. 2A). The ratio of immobilized EGFR was not as high as ˜61% despite treatment with a sufficient amount of antibody capable of immobilizing not only the intentionally expressed EGFR but also the endogenously expressed EGFR (FIG. 2D). In general, the distance between the cell membrane and the cover slip surface reaching 50 nm or more is about 30 nm from the cover slip surface, and the height of the EGFR is estimated to be about 15 nm from the cover slip surface. It was judged that EGFR was not sufficiently immobilized because it could only physically bind to only some surfaces in the cells adhered to the coverslip. Therefore, we wanted to increase the height from the coverslip by treating one additional antibody (secondary antibody) between neutravidin and EGFR specific antibodies (FIG. 2B). The expected height using the secondary antibody was about ˜45 nm, resulting in immobilization of> 97.3% of EGFR in COS7 cells that adhered well to the coverslip (FIG. 2D). In addition, no significant improvement in the immobilization rate of EGFR was observed even when the anticipated height was increased to ˜60 nm by introducing a tertiary antibody (FIG. 2C, D). The amount of EGFR specific antibody coated on the coverslip utilizing secondary antibodies was sufficient to immobilize all EGFR at a wide range of expression levels in COS7 cells (FIG. 3). In addition, it was confirmed that EGFR is immobilized to a sufficient level similarly to various types of EGFR specific antibodies used for immobilization using various antibodies that bind to different sites of EGFR such as mAb 199.12, mAb R-1, mAb 528, etc. (FIGS. 4A-C). In addition, the EGFR (SNAP-EGFR) with the SNAP tag coupled to the N-terminus of EGFR and the EGFR (mEos3.2-EGFR) with the mEos3.2 protein bound to the SNAP tag-specific and mEos3.2 protein-specific antibodies, respectively. Immobilization efficiency for the present invention was examined. SNAP-EGFR and mEos3.2-EGFR showed almost complete immobilization efficiency against SNAP tag-specific and mEos3.2 protein-specific antibodies, respectively, but EGFR-mEos3.2 with mEos3.2 linked to the C terminus of EGFR .2 unaffected by protein specific antibodies (FIGS. 4D-F). In addition, it was confirmed that more than 90% EGFR is effectively immobilized in different cell lines, such as HEK293 cells, HeLa cells and CHO-K1 cells (Fig. 5). Through the above results, it can be seen that the difference in the binding environment of the antibody or the microenvironment of the cell membrane does not significantly affect the efficiency of immobilization if the height of the antibody is satisfied from the surface of the coverslip to the cell.
공동 면역 고정법을 시행하는 데 있어서 가장 큰 우려는 살아있는 세포의 세포막에서 미끼 단백질의 고정화가 다른 전체 막 단백질에 영향을 주지 않고 특이적으로 이루어지는지에 대한 것이었다. 만약 커버슬립에 코팅된 EGFR 특이적인 항체가 세포막 위의 다른 막 단백질을 비특이적으로 심각한 수준으로 고정화시키게 되면 먹이 단백질의 고정화 부분모집단 비율의 증가가 고정화된 미끼 단백질이 아닌 다른 비특이적으로 고정화된 막 단백질과의 상호작용에 의한 것일 수 있기 때문이다. 따라서 우리는 EGFR 특이적인 항체를 이용하여 특정 항체의 처리가 표적이 아닌 다른 막 단백질의 고정화를 유도할 수 있는지 확인하였다.The greatest concern in implementing co-immunoimmobilization was whether immobilization of bait proteins in the cell membranes of living cells was specific without affecting other whole membrane proteins. If EGFR-specific antibodies coated on coverslips immobilize other membrane proteins on the cell membrane to nonspecific and severe levels, an increase in the immobilized subpopulation ratio of the feed protein may be associated with nonspecific immobilized membrane proteins other than immobilized bait proteins. This may be due to the interaction of. Therefore, we used EGFR-specific antibodies to determine whether treatment with specific antibodies could induce immobilization of membrane proteins other than the target.
먼저 EGFR과 물리적으로 상호작용하지 않는다고 알려진 beta2 adrenergic receptor (β2-AR)를 선택하여(도 6a) SNAP tag을 기반으로 CF660R 형광염료로 레이블링된 EGFR과 mEos3.2 형광단백질로 레이블링된 β2-AR를 함께 발현시킨 동일 COS7 세포에서 EGFR 특이적인 항체 처리 전후 조건에서 동시에 두 가지 막 단백질의 움직임을 관찰하였다(도 6b). 경로 데이터로부터 고정화된 부분모집단의 양을 정량화하기 위해서 확산에 대한 관계식 MSD = 4DΔt (0 < Δt < 780 ms)를 경로 데이터에 적용하여 확산계수를 계산하였으며, 고정화를 정의하기 위한 확산계수의 기준은 위치 오류를 바탕으로 결정되었다(도 6c). 실제로는 고정되어 있는 경로가 마치 움직이는 것처럼 나타날 때 평균 확산계수 σ^2/t로부터 위치 오류 σ를 도출할 수 있으므로 이를 이용하여 CF660R 염료와 mEos3.2 단백질의 위치 오류를 추정함으로써 각 단백질의 부분모집단을 구분짓는 확산계수 기준을 정의하였다. SNAP-EGFR이 EGFR 특이적인 항체에 의해 완전히 고정화되어 약 98.2%의 고정화된 부분모집단의 비율을 보이는 동안 β2-AR의 고정화 비율에는 유의미한 변화가 나타나지 않았다(도 6d). 비록 β2-AR의 움직이는 부분모집단의 확산계수가 ~13.4% 정도로 약간 감소하였으나, 이는 세포막과 커버슬립 사이의 혼잡도가 증가했기 때문으로 추측되며, 이와 같은 감소는 고정화된 부분모집단의 비율을 정량하는 데 있어서는 미미한 영향을 보였다.First, the beta2 adrenergic receptor (β2-AR), which is known to not physically interact with EGFR, was selected (Fig. 6a) to select EGFR labeled with the CF660R fluorescent dye and β2-AR labeled with the mEos3.2 fluorescent protein based on the SNAP tag. In the same COS7 cells expressed together, the movement of the two membrane proteins was simultaneously observed under conditions before and after EGFR specific antibody treatment (FIG. 6B). To quantify the amount of subpopulations immobilized from the path data, the diffusion coefficient MSD = 4DΔt (0 <Δt <780 ms) was applied to the path data and the diffusion coefficient was calculated. The determination was made based on the location error (FIG. 6C). In reality, when the fixed path appears to be moving, the position error σ can be derived from the average diffusion coefficient σ ^ 2 / t, which is used to estimate the position error of the CF660R dye and the mEos3.2 protein. The diffusion coefficient criterion that distinguishes is defined. There was no significant change in the immobilization rate of β2-AR while SNAP-EGFR was completely immobilized by the EGFR specific antibody showing a ratio of approximately 98.2% immobilized subpopulation (FIG. 6D). Although the diffusion coefficient of the β2-AR subpopulations decreased slightly by ~ 13.4%, this is presumably due to the increased congestion between the cell membrane and the coverslip, and this decrease was used to quantify the proportion of immobilized subpopulations. There was a slight effect.
우리는 추가적으로 EGFR에 특이적인 고정화가 다른 막 단백질에 미치는 영향도 알아보고자 하였다. 세포막에 특이적으로 위치할 수 있는 PMT(plasma membrane targeting) 신호 펩티드와 ErbB2 단백질 및 ErbB3 단백질, 그리고 인슐린 수용체 단백질(InsR)에 mEos3,2 형광 단백질을 표지한 후 EGFR 특이적 항체에 의한 고정화 비율을 측정하였다(도 6e). EGFR 특이적 항체를 처리하였을 때 EGFR의 고정화 비율이 90%가 넘는데 비해, PMT, ErbB3, InsR 및 β2-AR에서는 항체 처리 전후에 유의미한 변화가 관찰되지 않았다. 다만 ErbB2 단백질의 경우 평균적으로 약 ~8.7%의 고정화 효율이 증가하였는데 이는 유의미한 수준의 ErbB2가 EGFR과 상호작용을 하기 때문으로 해석된다. EGFR과 상호작용에 의해 유도된 고정화 비율의 증가를 최소화하고 EGFR 특이적인 항체에 의한 비특이적 고정화 비율만을 효과적으로 확인하기 위해서 내재된 EGFR의 발현량에 비해 이러한 막 단백질들을 과발현시켜서 측정을 진행하였으며, 이 중에서 오직 ErbB2와 ErbB3만이 EGFR과 상호작용한다고 알려져 있다는 점에서 ErbB3에 비해서 ErbB2이 EGFR과 상호작용을 통해 형성하는 이종이합체가 특히 강할 것으로 추측할 수 있다. 이러한 결과는 오로지 ErbB2 단백질만이 세포밖 영역이 펼쳐져 있는 구조를 갖고 있다는 기존의 구조 특성에 대한 연구결과와도 일맥상통한다.We also wanted to investigate the effect of EGFR-specific immobilization on other membrane proteins. Labeling of mEos3,2 fluorescent protein on PMT (plasma membrane targeting) signal peptide, ErbB2 protein and ErbB3 protein, and insulin receptor protein (InsR), which can be located on the cell membrane, and immobilization rate by EGFR specific antibody Measurement was made (FIG. 6E). There was no significant change in PMT, ErbB3, InsR and β2-AR before and after antibody treatment, whereas the EGFR immobilization rate was greater than 90% when treated with EGFR specific antibodies. However, in the case of the ErbB2 protein, the immobilization efficiency increased by ~ 8.7% on average, which is interpreted as a significant level of ErbB2 interacts with EGFR. In order to minimize the increase in the immobilization rate induced by the interaction with EGFR and to effectively identify only the nonspecific immobilization rate by the EGFR specific antibody, the expression was performed by overexpressing these membrane proteins relative to the expression level of the intrinsic EGFR. Since only ErbB2 and ErbB3 are known to interact with EGFR, it can be inferred that the heterodimers formed by ErbB2 by interacting with EGFR are particularly strong compared to ErbB3. These findings are in line with previous studies on the structural characteristics of which only the ErbB2 protein has an extended structure of extracellular regions.
실험예Experimental Example 3. 살아있는 단일 세포에서  3. In live single cells EGFREGFR 전- I'm- 이합체화에On dimerization 대한  About 해리상수의Dissociation constant 측정 Measure
EGFR의 전-동종 이합체(리간드 독립적인 이합체)에 대한 해리상수를 측정하기 위해 SNAP-EGFR과 EGFR-mEos3.2가 함께 발현된 세포에 SNAP tag 특이적인 항체를 처리하여 SNAP-EGFR만을 특이적으로 고정화하였다(도 7a). SNAP tag 대신 다른 단백질 tag이나 형광 단백질 또한 사용될 수 있지만 높은 밀도의 경로 데이터를 얻고 완전한 SNAP-EGFR의 고정화를 확인하기 위하여 SNAP tag을 사용하였다. EGFR의 전-동종 이합체화를 유사 1차 반응으로 가정하기 위해서는 SNAP-EGFR과 EGFR-mEos3.2의 발현 비율이 중요하기 때문에 EGFR-mEos3.2를 낮은 수준으로 안정적으로 발현하는 COS7 세포에 SNAP-EGFR을 과발현하였다. 그런 다음, SNAP tag 특이적 항체를 처리하기 전후의 조건에서 CF660R 형광 염료가 레이블링된 SNAP-EGFR과 EGFR-mEos3.2를 추적하여 각각의 경로 데이터를 얻었다(도 7b). SNAP-EGFR의 확산계수 분포도는 움직이는 상태의 경로들이 항체 처리 후에 완전히 고정화된 상태로 전환된 것(~95.2%)을 보여주는 반면 EGFR-mEos3.2는 약 ~22.7%의 부분적인 고정화된 상태로의 전환만을 보여주었다(도 7c-e). 여기에서 증가된 EGFR-mEos3.2의 고정화된 부분모집단의 비율은 결국 EGFR 전-동종 이합체의 양을 나타내는 것으로 볼 수 있다. 또한 단분자 수준에서 고정화된 SNAP-EGFR과 EGFR-mEos3.2의 경로에서 일시적으로 고정화 되는 위치가 정확히 겹쳐지는 것으로 보아 EGFR-mEos3.2의 일시적 고정화는 SNAP-EGFR과 이합체를 형성하기 때문인 것으로 생각할 수 있다(도 8).In order to measure the dissociation constant for all-homologous dimers of EGFR (ligand independent dimers), SNAP tag-specific antibodies were treated to cells expressing SNAP-EGFR and EGFR-mEos3.2 to specifically target SNAP-EGFR only. Immobilized (FIG. 7A). Other protein tags or fluorescent proteins may also be used instead of SNAP tags, but SNAP tags were used to obtain high-density pathway data and to confirm complete SNAP-EGFR immobilization. In order to assume the pre-homogenous dimerization of EGFR as a pseudo-first response, the expression ratio of SNAP-EGFR and EGFR-mEos3.2 is important, so SNAP- in COS7 cells stably expressing low levels of EGFR-mEos3.2 are important. EGFR overexpressed. Next, SNAP-EGFR and EGFR-mEos3.2 labeled CF660R fluorescent dyes were traced under conditions before and after treatment with SNAP tag specific antibodies to obtain respective pathway data (FIG. 7B). The diffusion coefficient distribution of SNAP-EGFR shows that the moving pathways are fully immobilized (~ 95.2%) after antibody treatment, whereas EGFR-mEos3.2 is approximately 22.7% partially immobilized. Only conversion was shown (FIG. 7C-E). The increased proportion of immobilized subpopulations of EGFR-mEos3.2 can be seen here to eventually indicate the amount of EGFR pre-homogenous dimers. In addition, since the position of the temporary immobilization in the pathway of immobilized SNAP-EGFR and EGFR-mEos3.2 overlaps precisely, the temporary immobilization of EGFR-mEos3.2 forms a dimer with SNAP-EGFR. May be (FIG. 8).
다음으로 표면의 총 형광 신호를 단분자 신호로 나눔으로써 COS7 세포 표면에서 발현되어 있는 SNAP-EGFR의 농도를 결정하였다(도 7f). 경로를 추적하기 전에 CF660R 형광 염료로 레이블링된 SNAP-EGFR의 전반사 형광 이미지를 얻었으며 여기에서 총 형광 신호의 세기가 바로 COS7 세포 표면에서의 SNAP-EGFR의 전체량이 된다. 모든 경로 추적 단계가 완료된 후 광탈색을 통해 SNAP 단백질이 개별 분자 수준에서의 관찰이 가능해질 때까지 형광 신호를 끈 다음 전반사 형광 이미지를 다시 얻었다. 이중에서도 특히 한 단계의 광탈색이 되는 것을 통해 단일 분자의 형광 신호를 골랐으며 이들의 평균을 취함으로써 한 개의 CF660R 형광 염료로부터 발생하는 단분자 형광 신호를 결정하였다. 추가적으로 CF660R 형광이 활성화되지 않아 고정화는 되었지만 탐지되지 않은 SNAP-EGFR의 비율을 고려하여 전체 SNAP-EGFR의 개수를 교정하였다(도 9). 이렇게 계산된 SNAP-EGFR의 농도는 ~650±54 molecules/μm2로 나타났다. 막 단백질은 2차원의 세포막 위에서 좌우로만 확산하기 때문에 세포막에서의 몰 농도의 정의가 명확하지 않기 때문에 기존에 사용하는 몰 농도 대신 2차원의 밀도로 계산되는 표기법을 사용하였다. SNAP-EGFR의 농도가 증가할수록 EGFR-mEos3.2가 고정화된 SNAP-EGFR과 만날 확률이 증가하게 되므로 따라서 이를 확인하기 위해 SNAP-EGFR의 발현 수준에 따른 EGFR-mEos3.2의 고정화 비율의 경향성을 분석하였으며 수학식 5에서 보여지는 관계와도 마찬가지로 SNAP-EGFR의 발현량과 EGFR-mEos3.2의 고정화된 SNAP-EGFR에 대한 비결합/결합 비율 사이의 선형 비례관계를 관찰할 수 있었다(도 10).Next, the concentration of SNAP-EGFR expressed on the surface of the COS7 cell was determined by dividing the total fluorescence signal on the surface by a single molecule signal (FIG. 7F). Before tracking the route, a total reflection fluorescence image of SNAP-EGFR labeled with CF660R fluorescent dye was obtained, where the intensity of the total fluorescence signal is the total amount of SNAP-EGFR at the surface of the COS7 cell. After all the path tracing steps were completed, the photobleaching turned off the fluorescence signal until the SNAP protein could be observed at the individual molecular level, and the total reflection fluorescence image was taken again. In particular, one step of photobleaching was used to select fluorescence signals of a single molecule, and their average was taken to determine a single molecule fluorescence signal generated from one CF660R fluorescent dye. Additionally, the total number of SNAP-EGFR was corrected in consideration of the ratio of SNAP-EGFR that was immobilized but not detected because CF660R fluorescence was not activated (FIG. 9). The calculated SNAP-EGFR concentration was 650 ± 54 molecules / μm 2 . Because membrane proteins only diffuse from side to side on two-dimensional cell membranes, the definition of molar concentration in the cell membranes is not clear. As the concentration of SNAP-EGFR increases, the probability of EGFR-mEos3.2 encounters the immobilized SNAP-EGFR increases. Therefore, in order to confirm this, the tendency of the immobilization ratio of EGFR-mEos3.2 according to the expression level of SNAP-EGFR is increased. As in the relationship shown in Equation 5, a linear proportional relationship between the expression level of SNAP-EGFR and the non-binding / binding ratio of EGFR-mEos3.2 to immobilized SNAP-EGFR was observed (FIG. 10). .
위에서 구한 수치들을 수학식 4에 대입한 결과 EGFR의 전-동종 이합체의 해리상수는 ~2727±126 molecules/μm2로 계산되었으며 이 수치값은 SNAP-EGFR의 발현량이 변하더라도 일정하게 나타나는 것을 확인하였다(도 7g). EGFR의 전-동종 이합체에 대한 결합 양상을 확인하기 위해 측정된 결과값들을 결합 플롯에 대입했으며 이를 통해 COS7 세포에서 측정된 범위의 발현 수준 안에서 EGFR은 협동성이 없는 단순한 이합체만을 형성하는 것을 확인하였다(도 7h). 또한 HEK293 세포, HeLa 세포 및 CHO-K1 세포에서 EGFR의 전-이합체의 해리상수를 측정하였을 때 모두 COS7 세포와 유의미한 차이가 없는 유사한 값이 측정되었다(도 7i).As a result of substituting the above equations into Equation 4, the dissociation constant of all-homodimer of EGFR was calculated to be ~ 2727 ± 126 molecules / μm 2 , which was found to be constant even when the expression level of SNAP-EGFR was changed. (Figure 7g). In order to confirm the binding pattern of all-homologous dimers of EGFR, the measured values were substituted into the binding plot, and it was confirmed that within the range of expression levels measured in COS7 cells, EGFR formed only simple dimers without cooperation. 7h). In addition, when measuring the dissociation constant of the pre-dimer of EGFR in HEK293 cells, HeLa cells and CHO-K1 cells, all of the similar values without significant difference was measured (FIG. 7I).
실험예 4. 리간드 의존적/독립적인 일시적인 EGFR의 이합체화Experimental Example 4 Dimerization of Ligand-dependent / Independent Transient EGFR
다음으로 우리는 EGFR의 리간드인 EGF의 유무에 따른 EGFR의 이합체화를 정량적으로 분석하였다. 비록 SNAP-EGFR을 고정화하기 위해 사용되는 SNAP tag 특이적인 항체가 EGF의 결합을 방해하지는 않지만 EGFR이 고정화된 상태에 있더라도 EGF의 처리에 의해 인산화가 되는지 관찰함으로써 고정화에 의한 생리학적 영향이 없음을 확인하고자 하였다. mEos3.2 형광 단백질에 특이적 항체가 코팅되어 있는 커버슬립 위에 안착되어 있는 mEos3.2-EGFR이 발현되어 있는 세포에 EGF를 처리한 후 용해시켜 커버슬립 표면에 고정화된 mEos3.2-EGFR만을 남겼다. EGFR의 Y1068번 아미노산의 인산화를 표적으로 하는 항체를 이용하여 남아있는 EGFR에 표지하였을 때, 기본 인산화 수준과 비교하여 EGF 처리에 의한 고정화된 mEos3.2-EGFR의 유의미한 수준의 인산화 정도를 확인할 수 있었다(도 11).Next, we quantitatively analyzed the dimerization of EGFR with and without EGF, the ligand of EGFR. Although the SNAP tag-specific antibody used to immobilize SNAP-EGFR does not interfere with EGF binding, it is confirmed that there is no physiological effect by immobilization by observing that phosphorylation is performed by treatment of EGF even when EGFR is immobilized. Was intended. EGF treatment was performed on cells expressing mEos3.2-EGFR on the coverslip coated with a specific antibody coated on mEos3.2 fluorescent protein, followed by lysis, leaving only mEos3.2-EGFR immobilized on the coverslip surface. . When the remaining EGFR was labeled with an antibody that targets the phosphorylation of amino acid Y1068 of EGFR, the level of phosphorylation of immobilized mEos3.2-EGFR by EGF treatment was compared with that of the basic phosphorylation level. (FIG. 11).
SNAP-EGFR과 EGFR-mEos3.2가 함께 발현된 COS7 세포를 4시간 동안 굶긴 다음 공동 면역 고정법을 이용하여 EGF 유무에 따른 EGFR의 이합체화에 대한 해리상수를 측정하였다. 유사 1차 반응의 조건을 충족시키기 위해 SNAP-EGFR의 발현 수준은 EGFR-mEos3.2의 발현 수준보다 최소 10배 이상 높도록 유지하였다. EGF가 없는 EGFR 전-이합체의 해리상수는 ~7244 molecules/μm2로 측정되었으며, EGF에 의해 형성된 EGFR 이합체의 해리상수는 ~369 molecules/μm2로 측정되었다. EGF의 처리는 해리상수를 20배 가량 차이나도록 함으로써 EGF에 의한 EGFR의 인산화를 유의미한 수준으로 증가시키는 데 기여했을 것으로 추측할 수 있다. 또한 세포가 굶주린 환경에서 EGFR 전-이합체의 해리상수는 세포가 성장하는 일반적인 조건(10% FBS)에서의 해리상수보다(도 7g) 약 2.6배 정도 더 높은 값을 갖는 것을 확인할 수 있었으며 이는 다른 간접적인 요소들이 EGFR의 이합체의 형성에 영향을 미칠 수 있다는 것을 의미한다.COS7 cells expressing both SNAP-EGFR and EGFR-mEos3.2 were starved for 4 hours, and then dissociation constants for dimerization of EGFR with or without EGF were measured using co-immuno immobilization. The expression level of SNAP-EGFR was maintained at least 10 times higher than the expression level of EGFR-mEos3.2 to meet the conditions of the similar primary response. The dissociation constant of EGFR pre-dimer without EGF was determined to be ~ 7244 molecules / μm 2 , and the dissociation constant of EGFR dimer formed by EGF was measured to be ~ 369 molecules / μm 2 . It can be speculated that the treatment of EGF may have contributed to a significant increase in the phosphorylation of EGFR by EGF by about 20 times the dissociation constant. In addition, the dissociation constant of the EGFR predimer in the cell-hungry environment was found to be about 2.6 times higher than the dissociation constant under normal conditions (10% FBS) where the cells grow (Fig. 7g), which is indirect. It means that phosphorus elements can affect the formation of dimers of EGFR.
EGFR에 대한 다른 인공적인 리간드들의 결합이 EGFR의 이합체화에 미치는 영향을 더 탐구하기 위하여 EGFR의 세포내 부위에 있는 ATP 결합 포켓을 표적하는 티로신 인산화 효소 저해제(Tyrosine kinase inhibitor, TKI)인 Erlotinib과 Lapatinib를 처리하였을 때 EGFR 이합체화의 해리상수를 측정하였다(도 12a). 그 결과 Erlotinib과 Lapatinib의 결합이 EGFR의 인산화를 저해함에도 불구하고 두 TKI 모두 EGF가 없는 환경에서도 EGFR 이합체화의 경향성을 증가시킴을 확인하였다. 이는 이러한 저해제의 결합이 EGFR의 세포 안쪽 부위의 변화를 통해 이합체화 가능성을 증가시킨 결과로 해석할 수 있다(도 12b). 흥미롭게도 Erlotinib은 EGF가 처리된 환경에서는 해리상수에 변화가 없었지만 Lapatinib은 EGF의 처리에도 불구하고 EGFR 이합체의 형성을 유의미하게 저해함을 확인하였다. 이러한 결과를 통해 Lapatinib의 결합이 EGFR의 구조적 형태를 EGFR 이합체의 안정성이 낮아지는 방향으로 유도하여 이합체의 형성이 불리한 형태로 변화됨을 알 수 있으며 이는 이전의 연구들과도 일관된 결과이다.To further explore the effects of binding of other artificial ligands to EGFR on dimerization of EGFR, Erlotinib and Lapatinib, tyrosine kinase inhibitors (TKIs) targeting ATP binding pockets in the intracellular site of EGFR The dissociation constant of EGFR dimerization was measured when was treated (Fig. 12a). As a result, although the binding of Erlotinib and Lapatinib inhibited EGFR phosphorylation, both TKIs increased the tendency of EGFR dimerization even in the absence of EGF. This can be interpreted as the result of the binding of these inhibitors increased the possibility of dimerization through the change of the inner region of the EGFR (Fig. 12b). Interestingly, Erlotinib showed no change in dissociation constant in EGF-treated environment, but Lapatinib significantly inhibited the formation of EGFR dimers despite EGF treatment. These results indicate that the binding of Lapatinib induces the structural form of EGFR in the direction of lowering the stability of EGFR dimers, thereby changing the formation of the dimers into an unfavorable form, which is consistent with previous studies.
실험예 5. 세포막의 미세환경에 다른 EGFR의 이합체화Experimental Example 5 Dimerization of Different EGFR to Microenvironment of Cell Membrane
세포막의 골격인 액틴을 분해하거나 세포막으로부터 콜레스테롤을 제거함으로써 세포막 주변의 미세환경의 변화에 의한 EGFR의 이합체화에 대한 간접적인 효과에 대해 알아보고자 하였다. 이전의 연구에서 세포막 아래에 형성되어 있는 액틴의 그물구조가 EGFR의 이합체화에 영향을 미칠 수 있다는 보고가 되어 있었기 때문에 이를 직접적으로 증명하고자 액틴 구조를 적당히 분해시키는 Latrunculin B를 처리한 후 EGFR의 이합체에 대한 해리상수를 측정하였다. 그 결과 Latrunculin B를 처리한 후의 EGFR 이합체의 해리상수는 증가하였으며 액틴 그물구조에 갇혀있던 EGFR이 풀려나게 됨으로써 EGFR의 움직임이 더 자유로워짐을 관찰하였다(도 13a). 따라서 EGF에 의한 EGFR의 이합체 형성은 자유롭게 움직이는 EGFR의 국소적인 갇힘 현상에 의해 큰 영향을 받음을 알 수 있다(도 13b).The aim of this study was to investigate the indirect effects of dimerization of EGFR by changing the microenvironment around the cell membrane by decomposing actin or removing cholesterol from the cell membrane. Previous studies have reported that the actin's net structure under the cell membrane can affect the dimerization of EGFR. To demonstrate this directly, the EGFR dimer after treatment with Latrunculin B, which degrades the actin structure appropriately, has been reported. The dissociation constant for was measured. As a result, the dissociation constant of the EGFR dimer after treatment with Latrunculin B was increased and the EGFR trapped in the actin network was released, thereby releasing the movement of the EGFR more freely (FIG. 13A). Therefore, it can be seen that the formation of dimers of EGFR by EGF is greatly influenced by the local trapping phenomenon of freely moving EGFR (FIG. 13B).
놀랍게도 Nystatin의 처리에 의한 콜레스테롤의 제거는 EGF 의존적인 EGFR 이합체뿐 아니라 EGF 독립적인 EGFR의 이합체의 해리상수에도 상당한 영향을 미쳤음을 확인하였다. 특히 EGF가 없는 상태에서 Nystatin의 처리에 의해 EGFR의 이합체의 해리상수는 ~1665 molecules/μm2 정도로 크게 감소하였는데 이 수치는 같은 조건 하에서 EGF에 의해 형성된 EGFR의 이합체의 해리상수와 비슷한 값이다(도 13c). 이 결과는 콜레스테롤의 제거가 EGFR의 이합체 형성 가능성을 높여주는 반면 EGFR의 이합체 형성에 중요하게 작용하던 EGF 자체의 기능은 완전히 없애버린다고 해석할 수 있다. 콜레스테롤이 많은 세포막 내 구역에서 EGF가 없는 경우 EGFR은 세포밖 부위가 접혀서 묶여있는 구조로 존재하는 경향이 있기 때문에 콜레스테롤의 제거가 EGFR의 접힌 구조를 펼치는 작용을 용이하게 하고 따라서 결과적으로 이합체 형성에 유리한 환경을 조성하는 것으로 추측된다. 그러나 동시에 세포막에서 콜레스테롤은 EGF에 의해 유도된 EGFR 이합체화를 안정화하는 기능을 할 것으로 보이기 때문에 Nystatin의 처리가 EGF에 의한 EGFR 이합체의 해리상수를 부분적으로 높이는 작용을 한 것으로 판단할 수 있다. EGFR 이합체화에 대한 콜레스테롤의 이러한 이중적인 특성은 리간드의 유무에 따른 EGFR의 이합체화의 차이를 더욱 크게 벌리게 되기 때문에 세포막에서 리간드에 의해 유도된 EGFR의 신호전달을 위한 지질 뗏목(lipid raft)의 중요한 기능을 보여준다고 할 수 있다. Surprisingly, the removal of cholesterol by Nystatin treatment significantly affected the dissociation constants of EGF-dependent EGFR dimers as well as EGF-dependent EGFR dimers. In particular, in the absence of EGF, the dissociation constant of EGFR dimers was greatly reduced to ~ 1665 molecules / μm 2 by Nystatin treatment, which was similar to the dissociation constant of dimers of EGFR formed by EGF under the same conditions (Fig. 13c). These results can be interpreted that the removal of cholesterol increases the possibility of dimer formation of EGFR, while completely eliminating the function of EGF itself, which was important for dimer formation of EGFR. In the absence of EGF in the area of cholesterol-rich cell membranes, EGFR tends to exist in a structure that is folded and bound to extracellular sites, thus eliminating cholesterol facilitates the action of unfolding the folded structure of EGFR and consequently favors dimer formation. It is assumed to create an environment. However, at the same time, cholesterol in the cell membrane seems to function to stabilize the EGFR dimerization induced by EGF, so Nystatin treatment may be considered to partially increase the dissociation constant of EGFR dimer by EGF. This dual nature of cholesterol to EGFR dimerization widens the difference in the dimerization of EGFR with and without ligand, which is an important function of the lipid raft for ligand-induced EGFR signaling in cell membranes. It can be said that.
우리는 초해상도 현미경을 활용한 단일 입자 추적 기술을 도입하여 공동 면역 고정화 현상을 감지함으로써 살아있는 단일 세포에서 막 단백질 간의 일시적인 상호작용을 정량적으로 분석하는 공동 면역 고정법을 개발하였다. 또한 이 방법 하에서 일시적인 상호작용에 대한 해리상수를 계산하기 위한 공식을 도출하였다. 비록 본 연구에서는 EGFR의 이합체 형성을 일시적인 상호작용에 대한 대표적인 모델로 제시하였지만 공동 면역 고정법의 활용은 약하고 일시적인 상호작용에만 제한되는 것은 아니다. 두 막 단백질의 결합강도가 미끼 단백질과 고정화를 위한 항체 사이의 결함 강도와 비슷한 정도로 강한 것이 아니라면 미끼 단백질의 발현 수준을 조절함으로써 강한 상호작용을 포함한 넓은 범위의 상호작용을 조사할 수 있다. 미끼 단백질의 최적 발현 수준은 상호작용의 해리상수에 따라 수학식 5를 통해 분석적으로 결정될 수 있다. 미끼 단백질을 고정화하는 효율은 측정 정확도의 관점에서 중요한 요소지만 굉장히 높게 과발현(백만개 이하의 수용체)된 세포에서도 이러한 고정화 효율이 크게 떨어지지 않는 것을 이미 확인한 바 있기 때문에(도 3) 대부분의 경우 충분한 정확성을 가지고 측정이 가능할 것으로 예상된다. 따라서 EGFR 전-이합체 보다 10배 이상 일시적으로 형성하는 상호작용까지도 공동 면역 고정법을 통해 분석이 가능하다.We have developed a co-immuno immobilization method that quantitatively analyzes transient interactions between membrane proteins in live single cells by detecting co-immuno immobilization by incorporating single particle tracking technology using super-resolution microscopy. Also under this method, a formula for calculating the dissociation constant for transient interactions was derived. Although the dimer formation of EGFR is a representative model for transient interactions in this study, the use of co-immunoimmobilization is not limited to weak and transient interactions. If the binding strength of the two membrane proteins is not as strong as the defect strength between the bait protein and the antibody for immobilization, a wide range of interactions, including strong interactions, can be investigated by controlling the expression level of the bait protein. The optimal expression level of the bait protein can be determined analytically through Equation 5 depending on the dissociation constant of the interaction. The efficiency of immobilizing the bait protein is an important factor in terms of measurement accuracy, but it has already been confirmed that even in highly overexpressed (less than one million receptor) cells, this immobilization efficiency does not drop significantly (FIG. 3). It is expected to be possible to measure. Therefore, even the interactions that form transiently 10 times or more than the EGFR pre-dimer can be analyzed by co-immuno immobilization.
우리는 공동 면역 고정법을 이용하여 EGFR의 전-동종 이합체화에 대해 분석하였다. 본 기술은 단일 세포에서 통계적으로 고정화된 부분모집단의 비율을 분석하는데 있어서 만 개 이하의 경로만으로도 충분하기 때문에 내재 막 단백질과 같은 낮은 발현 수준을 가진 단백질의 일시적인 상호작용도 측정이 가능하다. EGFR 항체의 Fab fragment에 Alexa fluor 647과 같은 형광 염료를 결합시켜서 EGFR을 레이블링하는 데 사용하면 별다른 발현 없이도 내재된 EGFR의 전-이합체화에 대한 해리상수를 측정할 수 있었으며 그 수치는 외부에서 발현시킨 EGFR-mEos3.2를 이용하여 측정한 해리상수 값과 유사하게 나타났다.We analyzed for pre-homogenous dimerization of EGFR using joint immunofixation. The technique allows for the measurement of transient interactions of proteins with low expression levels, such as endogenous membrane proteins, since fewer than 10,000 pathways are sufficient to analyze the proportion of statistically immobilized subpopulations in a single cell. By binding fluorescent dyes such as Alexa fluor 647 to Fab fragments of EGFR antibodies to label EGFR, the dissociation constant for pre-dimerization of intrinsic EGFR could be measured without any expression. It was similar to the dissociation constant measured using EGFR-mEos3.2.
우리는 EGFR 단량체들 사이의 상호작용을 유사 1차 반응으로 가정하고 해리상수를 계산하였으나 매우 높은 농도의 EGFR이 세포막에 발현되면 본 가정과는 다르게 높은 수준의 EGFR 복합체가 동시에 형성될 수 있다. 비록 EGFR 단량체 간의 해리상수가 EGFR의 발현 수준과는 무관함을 보여주긴 했으나(도 7g, h) 본 연구에서 측정한 범위를 넘어서서 극단적으로 높은 농도의 EGFR이 발현된 환경에서는 높은 수준의 저중합체가 형성될 수 있어서 측정된 결과값이 이로 인해 영향을 받을 수 있다.We calculated the dissociation constant assuming a similar first order interaction between the EGFR monomers, but if a very high concentration of EGFR is expressed in the cell membrane, a high level of EGFR complex can be formed simultaneously unlike this assumption. Although the dissociation constant between EGFR monomers was independent of the expression level of EGFR (Fig. 7g, h), high levels of oligomers were found in environments with extremely high concentrations of EGFR beyond the ranges measured in this study. Can be formed so that the measured results can be influenced thereby.
본 공동 면역 고정 기술은 일반적으로 살아있는 세포에서 넓은 범위의 결합강도를 가진 다양한 세포막 단백질의 상호작용에 적용될 수 있다. 다양한 생리학적 환경에서 막 단백질들의 정량적인 상호작용 특성을 밝혀내는 것은 살아있는 세포에서 막 단백질 간 상호작용이 갖는 역동적인 본질에 대한 심도 있는 이해를 위한 새로운 관점을 제시할 것이다.This joint immunofixation technique is generally applicable to the interaction of various cell membrane proteins with a wide range of binding strengths in living cells. Identifying the quantitative interaction characteristics of membrane proteins in various physiological environments will provide a new perspective for an in-depth understanding of the dynamic nature of membrane protein interactions in living cells.
재료 및 방법Materials and methods
1. 플라스미드, 항체, 시약1. Plasmids, Antibodies, Reagents
SNAP tag이 연결된 융합 단백질을 만들기 위해 pSNAPf 벡터(N9183S, New England Biolabs)로부터 SNAP tag 부분을 얻어 이를 pcDNA3.1/mEos3.2-EGFR 벡터에 넣음으로써 pcDNA3.1/SNAP tag-EGFR을 얻을 수 있었다. 사용된 모든 플라스미드는 다음 참고문헌 1(Kim, D.H. et al. Analysis of Interactions between the Epidermal Growth Factor Receptor and Soluble Ligands on the Basis of Single-Molecule Diffusivity in the Membrane of Living Cells. Angew . Chem . Int . Ed. Engl. 54, 7028-32 (2015))에서 확인할 수 있다. 항체와 시약의 출처는 다음과 같다: mAb 199.12 와 Alexa fluor 647 연결된 anti-mouse 항체는 Invitrogen에서 구매; mAb 528 과 mAb R-1 는 Santa Cruz에서 구매; Rabbit anti-Mouse IgG 와 biotin 연결된 EGFR 항체는 Thermo Scientific에서 구매; anti-mEos3.2 항체는 Badrilla에서 구매; anti-phosphorylated EGFR 항체(Y1068) 는 abcam에서 구매; the anti-actin 항체는 MP Biomedicals에서 구매; Erlotinib 과 lapatinib 은 Roche에서 구매; Nystatin 과 latrunculin B 는 Sigma Aldrich에서 구매하였다.PcDNA3.1 / SNAP tag-EGFR was obtained by obtaining the SNAP tag portion from the pSNAPf vector (N9183S, New England Biolabs) to create a fusion protein to which the SNAP tag was linked, and inserting it into the pcDNA3.1 / mEos3.2-EGFR vector. . All plasmids used are the following references 1 (Kim, DH et al. Analysis of Interactions between the Epidermal Growth Factor Receptor and Soluble Ligands on the Basis of Single-Molecule Diffusivity in the Membrane of Living Cells. Angew. Chem. Int. Ed Engl. 54 , 7028-32 (2015). The sources of antibodies and reagents are as follows: mAb 199.12 and Alexa fluor 647 linked anti-mouse antibodies are available from Invitrogen; mAb 528 and mAb R-1 are purchased from Santa Cruz; Rabbit anti-Mouse IgG and biotin-linked EGFR antibodies are available from Thermo Scientific; anti-mEos3.2 antibodies are available from Badrilla; anti-phosphorylated EGFR antibody (Y1068) was purchased from abcam; the anti-actin antibodies are available from MP Biomedicals; Erlotinib and lapatinib are purchased from Roche; Nystatin and latrunculin B were purchased from Sigma Aldrich.
2. 세포 배양 및 형질 전환2. Cell Culture and Transformation
COS7, HEK293, HeLa 세포는 American Type Culture Collection(ATCC)에서 구매하였고, 10 % FBS(Gibco)가 포함된 Dulbecco's modified Eagle medium(DMEM, Lonza)배지로 37°C와 5% CO2 그리고 95% 습도 환경에서 세포 배양하였다. 동일하게 ATCC에서 구매한 CHO-K1 세포는 DMEM과 F-12 배지(Thermo Scientific)를 1:1로 섞고 10% FBS가 추가된 배지로 37°C와 5% CO2 그리고 95% 습도 환경에서 세포 배양하였다. 모든 세포의 형질전환은 Lipofectamine LTX(Invitrogen)을 이용해 제조사의 지시에 따라 사용하였다.COS7, HEK293, and HeLa cells were purchased from the American Type Culture Collection (ATCC), Dulbecco's modified Eagle medium (DMEM, Lonza) medium containing 10% FBS (Gibco) at 37 ° C, 5% CO2 and 95% humidity. Cells were cultured. In the same way, CHO-K1 cells purchased from ATCC were cultured at 37 ° C, 5% CO2, and 95% humidity in a medium containing 1: 1 mixed DMEM with F-12 medium (Thermo Scientific) and 10% FBS. It was. All cells were transformed using Lipofectamine LTX (Invitrogen) according to the manufacturer's instructions.
3. 시료 준비3. Sample Preparation
커버슬립은 처음에 클로로포름/메탄올(50:50)로 24시간 동안 씻어주고, 그 후 에탄올에 넣어서 보관하였다. 커버슬립을 건조시킨 후에 플라즈마 챔버(Femto Science)에 1분 30초 동안 산화시키고, 메탄올에 탈이온수 4.5%, 아세트산 0.9%, 3 mercapto-pro-pyulrimethoxy silane(S10475, Fluorochem) 2.5%가 포함된 실레인용액에 넣어 4℃에서 약 9-12시간 동안 반응 시킨다. 처리된 커버슬립을 Phosphate Buffered Saline 용액으로 3회 세척 후, 50 μg/ml 농도로 fibronectin 용액에 희석된 maleimide 반응기가 활성화된 neutravidin 단백질(31007, Thermo Scientific)과 1시간 동안 상온에서 반응시키고, 세포를 안착시키기 전에 biotin이 결합된 goat anti-rabbit IgG H&L 항체(ab7089, abcam) 혹은 biotin이 결합된 goat anti-mouse IgG Fc 항체(A16088, Invitrogen)와 추가적으로 1시간 동안 반응시켜 주었다. 형광 이미징을 하는 동안 페놀레드로 인한 간섭을 없애고자, 세포는 커버슬립에 안착 후 페놀레드가 없는 10% serum이 포함된 DMEM(Thermo Scientific)로 계속 유지시켰다.The coverslips were first washed with chloroform / methanol (50:50) for 24 hours and then stored in ethanol. After drying, the coverslip was oxidized in a plasma chamber (Femto Science) for 1 minute and 30 seconds, and in methanol, a silane containing 4.5% of deionized water, 0.9% of acetic acid and 2.5% of 3 mercapto-pro-pyulrimethoxy silane (S10475, Fluorochem). Into the citation solution and reacted at 4 ℃ for about 9-12 hours. The treated coverslips were washed three times with a solution of Phosphate Buffered Saline, and then reacted with a activated maleimide reactor (31007, Thermo Scientific) at room temperature for 1 hour in a maleimide reactor diluted in fibronectin solution at a concentration of 50 μg / ml. The biotin conjugated goat anti-rabbit IgG H & L antibody (ab7089, abcam) or biotin conjugated goat anti-mouse IgG Fc antibody (A16088, Invitrogen) was further reacted for 1 hour prior to settlement. To eliminate phenol red interference during fluorescence imaging, cells were kept on DMEM (Thermo Scientific) containing 10% serum free of phenol red after seating on the coverslip.
4. 이미징을 위한 형광 염료 및 표지된 세포의 준비4. Preparation of Fluorescent Dye and Labeled Cells for Imaging
succinimidyl ester 반응기를 가진 CF660R(92134, biotium)는 디메틸포름아미드(DMF)에 있는 BG-NH2 (New England Biolabs)와 제조사의 지시에 따라 30°C에서 약 9-12시간 동안 반응시켰다. 반응 후 용매는 진공에서 증발시켰고, 생성물은 HPLC로 정제 후에 증류수에 녹였다.CF660R (92134, biotium) with succinimidyl ester reactor was reacted with BG-NH2 (New England Biolabs) in dimethylformamide (DMF) for about 9-12 hours at 30 ° C according to the manufacturer's instructions. After reaction the solvent was evaporated in vacuo and the product was dissolved in distilled water after purification by HPLC.
SNAP-EGFR이 발현된 세포는 EGFR에 형광 표지를 위해 기 준비된 BG-CF660R 혹은 SNAP-Surface Alexa Fluor 647(New England Biolabs)와 함께 반응시키고, 그 후 항체가 코팅된 커버슬립 위에 안착시켰다. Alexa flour 647를 이용한 형광 이미징의 경우에는 미디어에 추가적으로 탈산소제와 함께 환원제를 참고문헌 1과 같이 넣어준다.Cells expressing SNAP-EGFR were reacted with BG-CF660R or SNAP-Surface Alexa Fluor 647 (New England Biolabs), which were previously prepared for fluorescent labeling in EGFR, and then seated on an antibody-coated coverslip. In the case of fluorescence imaging using Alexa flour 647, a reductant is added to the media together with a deoxidant as shown in Ref.
5. 광학 설비 및 이미지 데이터의 확보와 처리5. Acquisition and processing of optical equipment and image data
형광 이미징은 XYZ축 자동화 스테이지가 장착된, 대물렌즈 기반의 TIRF 형광 현미경(IX-71, Olympus)에서 수행하였다. 실험에 사용된 레이저와 형광 필터는 참고문헌 1과 같이 사용하였다. 모든 형광 이미지는 전자증폭 결합소주 장치(EM-CCD/ iXon3 897, Andor Technology)를 사용해 얻어졌다. 보다 높은 배율의 이미지를 얻기 위해 현미경에 장착된 1.6x 증폭장치와 함께 1.43x의 튜브 렌즈를 사용하였다. 이미징을 하는 동안 살아있는 세포를 유지하기 위해 라이브셀 이미징 장치(Chamlide TC-A, Live cell instrument)를 사용하였다. 모든 장비의 조작과 이미징 데이터 확보는 MetaMorph (Molecular Devices)와 직접 작성한 MATLAB(The MathWorks) 플러그인을 사용해 이루어졌다. 대칭의 가우시안 함수를 이용한 비선형 최소 자승 근사법을 기반으로 하여, 원 이미지에서 다중 입자 추적을 할 수 있는 MATLAB 코드를 작성하였다. 이미지 프레임과 프레임 사이의 입자들 연결은 참고문헌 2(Jaqaman, K. et al. Robust single-particle tracking in live-cell time-lapse sequences. Nat. Methods 5, 695-702 (2008)) 에서 제시하는 단일 입자 추적 방법에 사용되는 u-track 프로그램으로 선형 할당 문제를 해결함으로써 가능해졌다. 그러나 연속된 프레임에서 추적 과정에서 사라지거나 두 개의 경로가 합쳐지는 경우에는 개별 입자의 추적을 즉시 종료하였다. 실험에 사용된 광학 부품의 조작과 이미징 데이터의 처리는 참고문헌 1과 같이 진행되었다.Fluorescence imaging was performed on an objective-based TIRF fluorescence microscope (IX-71, Olympus) equipped with an XYZ axis automated stage. The laser and fluorescence filter used in the experiment were used as in Reference 1. All fluorescence images were obtained using an electron amplification coupled soju device (EM-CCD / iXon3 897, Andor Technology). A tube lens of 1.43x was used with a 1.6x amplifier mounted on the microscope to obtain higher magnification images. Live cell imaging equipment (Chamlide TC-A, Live cell instrument) was used to maintain live cells during imaging. All instrument manipulation and imaging data was obtained using MetaMorph (Molecular Devices) and a hand-written MATLAB (The MathWorks) plug-in. Based on the nonlinear least-squares approximation using a symmetric Gaussian function, we developed a MATLAB code for multiple particle tracking in the original image. The linking of particles between the image frame and the frame is shown in Jaqaman, K. et al. Robust single-particle tracking in live-cell time-lapse sequences. Nat. Methods 5 , 695-702 (2008). The u-track program used in the single particle tracking method is made possible by solving the linear allocation problem. However, in case of disappearing during tracking in two consecutive frames or when two paths are merged, tracking of individual particles is terminated immediately. Manipulation of the optical components and processing of the imaging data used in the experiment proceeded as in Ref.
6. 해리상수의 계산6. Calculation of dissociation constant
확산계수는 입자가 2차원의 공간에서 보통의 확산현상을 하는 것을 가정하고 수학식 1과 수학식 2의 평균 자승의 변위(the mean squared displacements, MSD)를 사용하여 계산되었다.Diffusion coefficients were calculated using the mean squared displacements (MSD) of Equations 1 and 2, assuming that the particles had a normal diffusion phenomenon in two-dimensional space.
[수학식 1][Equation 1]
Figure PCTKR2017005544-appb-I000002
Figure PCTKR2017005544-appb-I000002
(MSD: 이동한 거리 제곱 평균(Mean Square Displacement), Δ: 표적 막 단백질 입자의 좌표 간 지연 시간, Δ는 자연수다, T: 하나의 궤적을 이루는 표적 막 단백질 입자의 좌표의 총 수, (x0, y0): 시작 위치, (xt,yt): 시간 t에서의 위치)(MSD: Mean Square Displacement, Δ: delay time between coordinates of target membrane protein particles, Δ is a natural number, T: total number of coordinates of target membrane protein particles forming one trajectory, (x 0 , y 0 ): starting position, (x t , y t ): position at time t)
[수학식 2][Equation 2]
MSD(Δ)= 4DΔ MSD (Δ) = 4DΔ
(D: 확산계수,Δ: 표적 막 단백질 입자의 좌표 간 지연 시간)(D: diffusion coefficient, Δ: delay time between coordinates of the target membrane protein particles)
'먹이' 단백질 R과 '미끼' 단백질 I 사이의 상호작용으로 인해 복합체 RI가 만들어질 때, 해리상수는 다음 수학식 3과 같이 정의된다.When the complex RI is created due to the interaction between the 'prey' protein R and the 'bait' protein I, the dissociation constant is defined as in Equation 3 below.
KD = [R][I] / [RI] … (수학식 3)K D = [R] [I] / [RI]. (Equation 3)
단, [R], [I], [RI]는 각각 개별 단백질 R, I, 복합체 RI의 농도를 나타낸다.However, [R], [I], and [RI] represent the concentrations of the individual proteins R, I, and complex RI, respectively.
막 단백질 I를 고정화시킨 후 막 단백질 R의 확산계수를 확률 밀도 함수로 측정하면 R과 I의 복합체 형성에 따라 R의 고정화 비율 역시 증가하게 된다. 이 고정화 비율을 ρR로 나타내면 해리상수는 다음 수학식 4과 같이 표현될 수 있다.After immobilization of membrane protein I, the diffusion coefficient of membrane protein R is measured as a probability density function, and the immobilization ratio of R increases as the complexes of R and I are formed. When the immobilization ratio is expressed by ρ R , the dissociation constant may be expressed as Equation 4 below.
KD = [R0](1-ρR)([I0]-[RI]) / [R0]ρR … (수학식 4)K D = [R 0 ] (1-ρ R ) ([I 0 ]-[RI]) / [R 0 ] ρ R. (Equation 4)
단, [R0]와 [I0]는 단백질 R과 I의 초기 농도이다.However, [R 0 ] and [I 0 ] are the initial concentrations of proteins R and I.
초기 I의 농도 [I0]가 복합체 RI의 농도 [RI]보다 훨씬 높은 조건을 가정하면 해리상수의 수식은 다음 수학식 5와 같이 단순화될 수 있다.Assuming that the initial concentration [I 0 ] is much higher than the concentration [RI] of the complex RI, the equation of the dissociation constant can be simplified as shown in Equation 5 below.
KD = (1-ρR)[I]0 / ρR … (수학식 5)K D = (1-ρ R ) [I] 0 / ρ R. (Equation 5)
따라서 해리상수는 I의 초기 농도인 [I0]와 I에 특이적인 항체 처리 후에 증가된 고정화 비율인 ρR의 두 값을 이용하여 계산될 수 있다.Therefore, the dissociation constant can be calculated using two values, [I 0 ], which is the initial concentration of I, and ρ R , which is an increased immobilization ratio after antibody treatment specific to I.
7. 막 단백질 발현 수준의 정량화7. Quantification of Membrane Protein Expression Levels
세포의 막 단백질과 함께 발현된 mEos3.2 형광 단백질은 488nm 파장의 레이저에 의해서, 그리고 막 단백질에 표지된 유기 형광 염료 CF660R 및 Alexa Flour 647은 642nm 파장의 레이저에 의해서 각각 조사되었다. 공간적인 편차를 최소화하기 위해 ImageJ 프로그램을 이용하여 동일한 세포에서 최소 다섯 곳의 위치와 세 장 이상의 이미지에서 배경 신호를 제거한 평균 형광 신호를 측정함으로써 총 발현 정도를 정량화하였다. 경로 추적을 위한 이미징이 종료된 후, 형광 분자들은 단분자 수준으로 관찰이 가능하도록 높은 파워의 레이저로 광탈색 과정을 진행하였다. 다섯 장 이상의 연속적인 이미지에 존재하는 형광 분자 중 단일 단계로 광탈색되는 분자들만을 골라서 그 평균 값을 단분자의 형광 신호 세기로 사용하였다. 단일 세포에서 발현된 특정한 막 단백질의 총 농도는 발현 정도에 따른 총 형광 세기를 단분자의 형광 세기로 나눔으로써 계산하였다.The mEos3.2 fluorescent protein expressed with the cell membrane protein was irradiated with a laser at 488 nm wavelength, and the organic fluorescent dyes CF660R and Alexa Flour 647 labeled with the membrane protein were irradiated with a laser at 642 nm wavelength, respectively. To minimize spatial variation, the total expression level was quantified by measuring the average fluorescence signal using at least five positions in the same cell and removing background signals from three or more images using the ImageJ program. After the imaging for path tracking was completed, the fluorescent molecules were subjected to photobleaching with a high power laser to observe at a single molecule level. Of the fluorescent molecules present in five or more consecutive images, only those molecules that were photobleached in a single step were selected and their average values were used as the fluorescence signal intensity of a single molecule. The total concentration of a particular membrane protein expressed in a single cell was calculated by dividing the total fluorescence intensity by the degree of expression by the fluorescence intensity of a single molecule.

Claims (16)

  1. 살아있는 세포에서 표적 막 단백질과 상기 표적 막 단백질에 대한 결합 파트너 막 단백질 간의 상호작용을 분석하는 방법에 있어서,A method for analyzing the interaction between a target membrane protein and a binding partner membrane protein for the target membrane protein in living cells,
    분석 대상 세포를 기재 상에 위치시키고,Place the cell of interest on the substrate,
    기재와 상기 표적 막 단백질 양자에 대한 결합능을 갖는 캡처 프로브를 분석 대상 세포에 처리하여 상기 표적 막 단백질을 기재 상에 고정시키고, A capture probe having binding capacity to both the substrate and the target membrane protein is treated with the cells of analysis to immobilize the target membrane protein on the substrate,
    상기 캡처 프로브의 처리 전과 후의 상기 결합 파트너 막 단백질의 확산계수 분포를 얻는 것을 포함하는Obtaining diffusion coefficient distribution of the binding partner membrane protein before and after treatment of the capture probe.
    막 단백질의 상호작용 분석 방법.Method for analyzing the interaction of membrane proteins.
  2. 제1항에 있어서,The method of claim 1,
    막 단백질은 내재성 막 단백질(integral membrane protein), 표재성 막 단백질(peripheral membrane protein), 막관통 단백질(transmembrane protein), 막 당단백질(membrane glycoprotein) 및 지질 공정 막 단백질(lipid anchored membrane protein)으로 이루어진 군으로부터 선택되는 것인 Membrane proteins are composed of integral membrane proteins, superipheral membrane proteins, transmembrane proteins, membrane glycoproteins, and lipid anchored membrane proteins. Selected from the group
    막 단백질의 상호작용 분석 방법.Method for analyzing the interaction of membrane proteins.
  3. 제1항에 있어서, The method of claim 1,
    막 단백질은 검출가능한 모이어티에 의해 레이블링되어 있는 것인 The membrane protein is labeled by a detectable moiety
    막 단백질의 상호작용 분석 방법.Method for analyzing the interaction of membrane proteins.
  4. 제1항에 있어서,The method of claim 1,
    막 단백질은 형광 단백질과의 융합 단백질이거나; 유기형광염료가 결합될 수 있는 태그와의 융합 단백질이면서 상기 융합 단백질의 태그에 형광물질이 레이블링되어 있거나; 형광물질이 결합되어 있고, 막 단백질에 대해 특이적으로 결합하는 프로브에 의해 레이블링되어 있는 상태인Membrane proteins are fusion proteins with fluorescent proteins; A fluorescent protein is labeled with a fusion protein with a tag to which an organic fluorescent dye can be bound, and a tag of the fusion protein; The fluorescent material is bound and labeled by a probe that specifically binds to the membrane protein.
    막 단백질의 상호작용 분석 방법.Method for analyzing the interaction of membrane proteins.
  5. 제1항에 있어서,The method of claim 1,
    상기 캡처 프로브는 항체, 항원-결합 단편, 어피바디, 또는 앱타머인The capture probe is an antibody, antigen-binding fragment, affibody, or aptamer
    막 단백질의 상호작용 분석 방법. Method for analyzing the interaction of membrane proteins.
  6. 제1항에 있어서, The method of claim 1,
    상기 기재는 뉴트라비딘, 스트렙타비딘, 또는 아비딘이 결합되어 있는 것인 The substrate is that neutravidin, streptavidin, or avidin is bound
    막 단백질의 상호작용 분석 방법.Method for analyzing the interaction of membrane proteins.
  7. 제1항에 있어서, The method of claim 1,
    상기 캡처 프로브는 항체이고, 비오틴이 결합되어 있는 것인The capture probe is an antibody, the biotin is bound
    막 단백질의 상호작용 분석 방법.Method for analyzing the interaction of membrane proteins.
  8. 제1항에 있어서, The method of claim 1,
    상기 결합 파트너 막 단백질의 확산계수는 단일 입자 추적(single particle tracking, SPT)을 통해 막 단백질의 세포막 내 이동 궤적을 탐지함으로써 얻는 것인The diffusion coefficient of the binding partner membrane protein is obtained by detecting the movement trajectory of the membrane protein in the cell membrane through single particle tracking (SPT).
    막 단백질의 상호작용 분석 방법.Method for analyzing the interaction of membrane proteins.
  9. 제8항에 있어서, The method of claim 8,
    상기 확산계수는 하기 수학식 1 및 수학식 2를 이용하여 얻는 것인 The diffusion coefficient is obtained by using the following Equations 1 and 2
    막 단백질의 상호작용 분석 방법.Method for analyzing the interaction of membrane proteins.
    [수학식 1][Equation 1]
    Figure PCTKR2017005544-appb-I000003
    Figure PCTKR2017005544-appb-I000003
    (MSD: 이동한 거리 제곱 평균(Mean Square Displacement), Δ: 표적 막 단백질 입자의 좌표 간 지연 시간, Δ는 자연수다, T: 하나의 궤적을 이루는 표적 막 단백질 입자의 좌표의 총 수, (x0, y0): 시작 위치, (xt,yt): 시간 t에서의 위치)(MSD: Mean Square Displacement, Δ: delay time between coordinates of target membrane protein particles, Δ is a natural number, T: total number of coordinates of target membrane protein particles forming one trajectory, (x 0 , y 0 ): starting position, (x t , y t ): position at time t)
    [수학식 2][Equation 2]
    MSD(Δ) = 4DΔ MSD (Δ) = 4DΔ
    (D: 확산계수,Δ: 표적 막 단백질 입자의 좌표 간 지연 시간)(D: diffusion coefficient, Δ: delay time between coordinates of the target membrane protein particles)
  10. 제1항에 있어서, The method of claim 1,
    상기 결합 파트너 막 단백질의 확산계수는 FRAP(Fluorescence recovery after photobleaching) 또는 FCS(Fluorescence Correlation Spectroscopy)에 의해 얻는 것인The diffusion coefficient of the binding partner membrane protein is obtained by Fluorescence recovery after photobleaching (FRAP) or Fluorescence Correlation Spectroscopy (FCS).
    막 단백질의 상호작용 분석 방법.Method for analyzing the interaction of membrane proteins.
  11. 제1항에 있어서, The method of claim 1,
    상기 캡처 프로브의 처리에 따른 확산계수 분포의 변화로부터 표적 막 단백질과 상기 표적 막 단백질에 대한 결합 파트너 막 단백질 간의 해리상수를 얻는 것을 추가로 포함하는 Further comprising obtaining a dissociation constant between the target membrane protein and the binding partner membrane protein for the target membrane protein from the change in diffusion coefficient distribution according to the treatment of the capture probe.
    막 단백질의 상호작용 분석 방법.Method for analyzing the interaction of membrane proteins.
  12. 제11항에 있어서, The method of claim 11,
    표적 막 단백질과 상기 표적 막 단백질에 대한 결합 파트너 막 단백질 간의 해리상수는 하기 [수학식 3], [수학식 4] 또는 [수학식 5]를 이용하여 얻는 것인The dissociation constant between the target membrane protein and the binding partner membrane protein for the target membrane protein is obtained using Equation 3, Equation 4 or Equation 5 below.
    막 단백질의 상호작용 분석 방법.Method for analyzing the interaction of membrane proteins.
    [수학식 3][Equation 3]
    KD = [R][I] / [RI] K D = [R] [I] / [RI]
    [수학식 4][Equation 4]
    KD = [R]0(1-ρR)([I]0-[RI]) / [R]0ρR K D = [R] 0 (1-ρ R ) ([I] 0- [RI]) / [R] 0 ρ R
    [수학식 5] [Equation 5]
    KD = (1-ρR)[I]0 / ρR K D = (1-ρ R ) [I] 0 / ρ R
    수학식 3에서, [I], [R], [RI]는 각각 표적 막 단백질의 농도, 상기 표적 막 단백질에 대한 결합 파트너 막 단백질의 농도 및 표적 막 단백질과 상기 표적 막 단백질에 대한 결합 파트너 막 단백질 간의 복합체의 농도를 나타내고, In Equation 3, [I], [R], and [RI] are the concentrations of the target membrane protein, the concentration of the binding partner membrane protein for the target membrane protein, and the binding partner membrane for the target membrane protein and the target membrane protein, respectively. Indicates the concentration of complexes between proteins,
    수학식 4에서 [R]0와 [I]0, 그리고 ρR은 각각 상호작용이 발생하기 이전의 초기 표적 막 단백질 및 결합 파트너 막 단백질의 농도, 캡쳐 프로브 처리 전과 비교하여 처리 후에 증가된 결합 파트너 막 단백질의 부분모집단 비율을 나타내며,In Equation 4, [R] 0 , [I] 0 , and ρ R are the concentrations of the initial target membrane protein and binding partner membrane protein before interaction, respectively, and the increased binding partner after treatment compared to before capture probe treatment. Represents a subset of membrane proteins,
    수학식 5에서, ρR은 캡처 프로브 처리 전과 비교하여 처리 후에서 증가된 결합 파트너 막 단백질의 부분모집단 비율이고, [I0]는 고정화된 표적 막 단백질의 절대 농도를 나타낸다.In Equation 5, ρ R is the subpopulation ratio of binding partner membrane protein increased after treatment compared to before the capture probe treatment, and [I 0 ] represents the absolute concentration of immobilized target membrane protein.
  13. 후보약물의 처리에 따른 살아있는 세포에서 표적 막 단백질과 상기 표적 막 단백질에 대한 결합 파트너 막 단백질 간의 상호작용의 변화를 분석하는 것을 포함하는 표적 막 단백질에 대한 후보약물의 스크리닝 방법에 있어서,A method for screening a candidate drug for a target membrane protein comprising analyzing a change in the interaction between the target membrane protein and a binding partner membrane protein for the target membrane protein in living cells following treatment of the candidate drug,
    분석 대상 세포를 기재 상에 위치시키고,Place the cell of interest on the substrate,
    상기 분석 대상 세포에 상기 표적 막 단백질에 영향을 미칠 것으로 예상되는 후보약물을 처리하고,Treating the cells of interest with candidate drugs expected to affect the target membrane protein,
    기재와 상기 표적 막 단백질 양자에 대한 결합능을 갖는 캡처 프로브를 처리하여 상기 표적 막 단백질을 기재 상에 고정시키고, Fix the target membrane protein on the substrate by treating a capture probe having binding capacity to both the substrate and the target membrane protein,
    상기 캡처 프로브의 처리 전과 후의 상기 결합 파트너 막 단백질의 확산계수 분포를 얻고,To obtain a distribution of diffusion coefficients of the binding partner membrane proteins before and after the capture probes,
    상기 결합 파트너 막 단백질의 확산계수 분포의 변화를 후보약물 무처리군에서의 캡처 프로브 처리 전과 후의 결합 파트너 막 단백질의 확산계수 분포의 변화와 비교하여 상기 표적 막 단백질과의 상호작용에 대해 영향을 미치는 후보약물을 선별하는 것을 포함하는The change in the distribution coefficient distribution of the binding partner membrane protein is compared with the change in the distribution coefficient distribution of the binding partner membrane protein before and after capture probe treatment in the candidate drug-free group to affect the interaction with the target membrane protein. Screening candidate drugs
    표적 막 단백질에 대한 후보약물의 스크리닝 방법.Screening method of candidate drug for target membrane protein.
  14. 제13항에 있어서, The method of claim 13,
    상기 후보약물은 화합물, 핵산, 당, 탄수화물, 지질, 펩티드 및 단백질로 이루어진 군으로부터 1종 이상 선택되는 것인 The candidate drug is one or more selected from the group consisting of compounds, nucleic acids, sugars, carbohydrates, lipids, peptides and proteins.
    표적 막 단백질에 대한 후보약물의 스크리닝 방법.Screening method of candidate drug for target membrane protein.
  15. 후보약물의 처리에 따른 살아있는 세포에서 표적 막 단백질과 상기 표적 막 단백질에 대한 결합 파트너 막 단백질 간의 상호작용의 변화를 분석하는 것을 포함하는 표적 막 단백질에 대한 후보약물의 스크리닝 방법에 있어서,A method for screening a candidate drug for a target membrane protein comprising analyzing a change in the interaction between the target membrane protein and a binding partner membrane protein for the target membrane protein in living cells following treatment of the candidate drug,
    분석 대상 세포를 기재 상에 위치시키고,Place the cell of interest on the substrate,
    상기 분석 대상 세포에 상기 표적 막 단백질에 영향을 미칠 것으로 예상되는 후보약물을 처리하고,Treating the cells of interest with candidate drugs expected to affect the target membrane protein,
    기재와 상기 표적 막 단백질 양자에 대한 결합능을 갖는 캡처 프로브를 처리하여 상기 표적 막 단백질을 기재 상에 고정시키고, Fix the target membrane protein on the substrate by treating a capture probe having binding capacity to both the substrate and the target membrane protein,
    상기 캡처 프로브의 처리 전과 후의 상기 결합 파트너 막 단백질의 표적 막 단백질에 대한 해리상수를 얻고,Dissociation constant for the target membrane protein of the binding partner membrane protein before and after treatment of the capture probe,
    후보약물 처리에 따른 결합 파트너 막 단백질의 해리상수의 변화를 후보약물 무처리군에서의 캡처 프로브 처리 전과 후의 결합 파트너 막 단백질 해리상수의 변화와 비교하여 상기 표적 막 단백질과의 상호작용에 대해 영향을 미치는 후보약물을 선별하는 것을 포함하는The change in the dissociation constant of the binding partner membrane protein following the candidate drug treatment was compared with the change in the binding partner membrane protein dissociation constant before and after the capture probe treatment in the candidate drug-free group to influence the interaction with the target membrane protein. Affecting the selection of candidate drugs
    표적 막 단백질에 대한 후보약물의 스크리닝 방법.Screening method of candidate drug for target membrane protein.
  16. 제15항에 있어서, The method of claim 15,
    상기 후보약물은 화합물, 핵산, 당, 탄수화물, 지질, 펩티드 및 단백질로 이루어진 군으로부터 1종 이상 선택되는 것인 The candidate drug is one or more selected from the group consisting of compounds, nucleic acids, sugars, carbohydrates, lipids, peptides and proteins.
    표적 막 단백질에 대한 후보약물의 스크리닝 방법.Screening method of candidate drug for target membrane protein.
PCT/KR2017/005544 2016-09-07 2017-05-26 Method for quantitatively measuring interaction between membrane proteins in living cells WO2018048063A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR1020160114740A KR101823990B1 (en) 2016-09-07 2016-09-07 A Method for Quantitative Analysis of Transient Interactions of Membrane Proteins in a Single Living Cell
KR10-2016-0114740 2016-09-07

Publications (1)

Publication Number Publication Date
WO2018048063A1 true WO2018048063A1 (en) 2018-03-15

Family

ID=61083455

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2017/005544 WO2018048063A1 (en) 2016-09-07 2017-05-26 Method for quantitatively measuring interaction between membrane proteins in living cells

Country Status (2)

Country Link
KR (1) KR101823990B1 (en)
WO (1) WO2018048063A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020069640A1 (en) * 2018-10-03 2020-04-09 福州大学 Method for measuring effective diffusion coefficient of biomolecule

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000017221A1 (en) * 1998-09-24 2000-03-30 Duke University Method of measuring protein-protein interactions in living cells
US20100323957A1 (en) * 2007-11-19 2010-12-23 The Regents Of The University Of California The Office Of The President Novel assay for inhibitors of egfr
KR101568565B1 (en) * 2014-01-06 2015-11-23 서울대학교산학협력단 Artificial cell membrane comprising supported lipid bilayer connected with probes having controllable mobility and method for analyzing interaction between molecules using the same
KR101600177B1 (en) * 2013-05-16 2016-03-07 포항공과대학교 산학협력단 Method of analyzing binding aspect of membrane protein in a living cell

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000017221A1 (en) * 1998-09-24 2000-03-30 Duke University Method of measuring protein-protein interactions in living cells
US20100323957A1 (en) * 2007-11-19 2010-12-23 The Regents Of The University Of California The Office Of The President Novel assay for inhibitors of egfr
KR101600177B1 (en) * 2013-05-16 2016-03-07 포항공과대학교 산학협력단 Method of analyzing binding aspect of membrane protein in a living cell
KR101568565B1 (en) * 2014-01-06 2015-11-23 서울대학교산학협력단 Artificial cell membrane comprising supported lipid bilayer connected with probes having controllable mobility and method for analyzing interaction between molecules using the same

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
QIAN ET AL.: "Single Particle Tracking. Analysis of Diffusion and Flow in Two-dimensional Systems", BIOPHYSICAL JOURNAL, vol. 60, October 1991 (1991-10-01), pages 910 - 921, XP055487337 *
ZHU ET AL.: "Analysis of Two-dimensional Dissociation Constant of Laterally Mobile Ceil Adhesion Molecules", BIOPHYSICAL JOURNAL, vol. 92, 1 February 2007 (2007-02-01), pages 1022 - 1034, XP055487350 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020069640A1 (en) * 2018-10-03 2020-04-09 福州大学 Method for measuring effective diffusion coefficient of biomolecule

Also Published As

Publication number Publication date
KR101823990B1 (en) 2018-01-31

Similar Documents

Publication Publication Date Title
Daly et al. Fluorescent ligands, antibodies, and proteins for the study of receptors
Sun et al. Monitoring protein interactions in living cells with fluorescence lifetime imaging microscopy
Han et al. Mechanistic studies of the genetically encoded fluorescent protein voltage probe ArcLight
Leuchowius et al. Flow cytometric in situ proximity ligation analyses of protein interactions and post‐translational modification of the epidermal growth factor receptor family
Alibhai et al. Automated fluorescence lifetime imaging plate reader and its application to Förster resonant energy transfer readout of Gag protein aggregation
Ivanusic et al. Investigation of membrane protein—protein interactions using correlative FRET-PLA
Pakhomov et al. Fluorescent protein Dendra2 as a ratiometric genetically encoded pH-sensor
EP2700947B1 (en) Method for analyzing protein-protein interaction on single-molecule level within the cellular environment
Erdelyi et al. Analyzing receptor assemblies in the cell membrane using fluorescence anisotropy imaging with TIRF microscopy
Jobin et al. Filamin A organizes γ‑aminobutyric acid type B receptors at the plasma membrane
WO2018048063A1 (en) Method for quantitatively measuring interaction between membrane proteins in living cells
Presley Imaging the secretory pathway: the past and future impact of live cell optical techniques
CN111164427A (en) Methods for measuring modulation of G protein-coupled receptor activity
Auriau et al. Gain of affinity for VEGF165 binding within the VEGFR2/NRP1 cellular complex detected by an HTRF-based binding assay
Pepperkok et al. System for quantitation of gene expression in single cells by computerized microimaging: Application to c-fos expression after microinjection of anti-casein kinase II antibody
Gidon et al. Studying the regulation of endosomal cAMP production in GPCR signaling
US20230183676A1 (en) Methods and systems for screening using microcapillary arrays
Nemec et al. Functional modulation of PTH1R activation and signalling by RAMP2
EP2518157A1 (en) Test Systems and methods for identifying a compound altering cellular DDR activity
Mahlandt et al. Cell-based optimization and characterization of genetically encoded location-based biosensors for Cdc42 or Rac activity
CN108645828B (en) Co-expression recombinant human protein biological activity and titer detection method based on multiple time-resolved fluorescence technologies
Sun et al. Probing the dynamics of growth factor receptor by single-molecule fluorescence imaging
Zhang et al. Visualizing directional Rab7 and TrkA cotrafficking in axons by pTIRF microscopy
Tovey et al. Confocal microscopy: theory and applications for cellular signaling
İşbilir Localization and Trafficking of CXCR4 and CXCR7

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: 17848948

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17848948

Country of ref document: EP

Kind code of ref document: A1