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WO2003008536A2 - Systeme de surveillance d'un processus intracellulaire et criblage in vivo de medicaments - Google Patents

Systeme de surveillance d'un processus intracellulaire et criblage in vivo de medicaments Download PDF

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Publication number
WO2003008536A2
WO2003008536A2 PCT/KR2002/001345 KR0201345W WO03008536A2 WO 2003008536 A2 WO2003008536 A2 WO 2003008536A2 KR 0201345 W KR0201345 W KR 0201345W WO 03008536 A2 WO03008536 A2 WO 03008536A2
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protein
cell
signal
trafficking
recombinant
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PCT/KR2002/001345
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English (en)
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Inhwan Hwang
Dae Heon Kim
Yong Jik Lee
Jing Bo Jin
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Ahram Biosystems Inc.
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Publication of WO2003008536A2 publication Critical patent/WO2003008536A2/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • 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
    • G01N33/5076—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 involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
    • 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
    • G01N33/6875—Nucleoproteins

Definitions

  • the present invention relates to the technology in cell molecular biology. More particularly, it relates to methods that make it possible to selectively observe intracellular trafficking of a selected protein and also to selectively screen new drugs that affect the expression or the intracellular trafficking of a selected protein in vivo. Especially, the present invention relates to a cell-based detection system that uses cells transformed to express a reporter protein labeled with a fluorescent protein.
  • Genomics and Proteomics researches become new leading trends to discover valuable genes and proteins from the vast amount of the primary information and also to elucidate and use the correlations among such bio-information.
  • technologies that can selectively and quickly sort and detect the detailed processes of the complex biological phenomena, as realized in the present invention.
  • Developing such technologies is one of the leading trends as found in various recent arts pertaining to the present invention.
  • the most important usage of the large amount of the useful information obtained from Genomics and Proteomics is for developing new drugs. Establishment of efficient and selective drug screening systems based on such useful bio-information is expected to play a major role in evolution of the biotechnology. Therefore, researches for developing new drug screening systems have been actively carried out recently.
  • the first is a protein- or a molecule-based method in which the effect of the drug candidate on the activity of an enzyme or a receptor critical in the induction mechanism of a disease is directly observed to screen the drug candidates.
  • the second is an organism-based method in which the effect of the drug candidate on the morphological or biochemical characteristics of an organism is observed to screen the drug candidates.
  • the last is a cell-based method in which the effect of the drug candidate on the morphology of the cell or subcellular organelles, and expression, trafficking, and metabolism of proteins in the cell is observed to screen the drug candidates.
  • the protein-based method has the advantage that the assay can be performed for a specific target protein. However, because the assay is conducted in vitro, it is not possible to examine various complex cellular factors.
  • the organism- or cell- based method can detect the composite changes of the organism or the cell induced by the drug candidates. But it also requires additional time-consuming experiments to confirm that the drug candidate specifically affects the target gene or protein.
  • fluorescent proteins such as green fluorescent protein (GFP), red fluorescent protein (REF) (Moris et al., 1974), and their derivatives (Evans, W098/21355) that can be expressed in the cell have been developed, and various researches have been reported using expression of these fluorescent proteins.
  • fluorescent proteins examples include observation of the gene expression and subcellular protein localization (Chalfie and Prasher, US5491084), visualization of the subcellular organelles (Kost et al., 1998), visualization of the protein trafficking in the secretory pathway (Kaether and Gerdes, 1995), and visualization of the protein expression pattern in a plant cell (Hu and Cheng, 1995) and in Drosophila embryo (Davis and Viestra, 1998).
  • the fluorescent proteins have been also used to develop new drug screening methods.
  • Examples include a method using mutated organisms transformed to express a modified GFP (Ward and Chalfie, W095/21191), a method using a cell transformed to express a fusion protein comprising a fluorescent protein and a transcription factor related to the activity of a cell surface receptor which regulates signal transduction (Harpold et al., US5401629), and a method using a cell transformed to express a fusion protein comprising a fluorescent protein and the active site of the protein kinase (Thastrup et al., WO96/23898).
  • the chlorophyll-binding proteins are transported to chloroplast, the nuclear localization signal domain (NLS domain) of SN40 is targeted to the nucleus (Goldfarb et al., 1986), the peroxisome targeting motif SKL is targeted to peroxisome (Davis and Viestra, 1998), and Fl- H + -ATPase is transported to mitochondria ( ⁇ iwa et al., 1999).
  • NLS domain nuclear localization signal domain
  • SKL is targeted to peroxisome
  • Fl- H + -ATPase is transported to mitochondria ( ⁇ iwa et al., 1999).
  • most of proteins in the cell are transported to specific subcellular organelles related to their functions.
  • PLC-Delta PH (Stauffer et al., 1998), AtPH, and FAPP1 (Dowler et al., 2000) domains are known to bind specifically to PI(3,4)P2, PI(3)P, and PI(4)P, respectively.
  • a fusion protein having the PH domain and GFP was shown to be translocated to the plasma membrane when expressed in a cell (Kost et al., 1998).
  • Selective drug screening methods can also be developed based on such detection scheme.
  • intracellular processes are very complicated because several thousands or several tens of thousands proteins are involved in these processes.
  • To develop a realistic method that can be used to detect each of the complicated intracellular processes selectively and systematically many different fusion proteins having fluorescent protein labels have to be constructed to enable selective detection of diverse intracellular processes, and the specific intracellular characteristics of these fusion proteins need to be elucidated.
  • the present invention provides:
  • the present invention also provides a systematic method for transforming a cell with the recombinant plasmid and expressing efficiently the reporter protein in the transformed cell. Furthermore, the present invention provides a detailed method for monitoring details of the intracellular distribution of the reporter protein in a living cell by observing the fluorescence image either continuously or step by step during the expression and trafficking processes. In addition to providing the efficient systematic method for measuring the spatial distribution specific to the expression and trafficking processes of a selected protein in the transformed cell, it is another objective of the present invention to provide a method for selectively screening drug candidates that affect a specific intracellular process in the transformed cell.
  • the present invention provides recombinant genes for diverse signal proteins that direct targeting to specific intracellular organelles such as nucleus, mitochondria, chloroplast, peroxisome, plasma membrane, endoplasmic reticulum, Golgi apparatus, storage vacuole, lytic vacuole, prevacuolar compartment, etc.
  • the present invention aims to provide an optimized overall procedure from the construction of the recombinant plasmids to the preparation of the transformed cells.
  • the present invention provides (1) the method for selectively screening chemicals that affect the intracellular trafficking and localization of a selected protein, (2) the method for screening chemicals that inhibit or enhance the transcription or translation process by monitoring the effects of the chemicals on the expression of the reporter protein, and (3) the method for screening cytotoxic chemicals by observing the effect of chemicals that cause deformation, damage, or disruption of subcellular cellular organelles.
  • FIG 1 shows schematic diagrams of the reporter proteins targeting to the subcellular organelles enclosed by membrane, which are classified as Group I proteins in the present invention.
  • Figure 2 shows fluorescence images showing the expression of the Group I reporter proteins in the cell, wherein (a) shows that AtOEP7:GFP is localized to the envelope of chloroplast, wherein the red fluorescent signal is the auto-fluorescence signal of chloroplast and the yellow fluorescence signal is an merged image of the auto-fluorescence signal of chloroplast and the green fluorescence signal of the reporter protein;
  • (b) is a photograph where the red auto-fluorescence of chloroplast in (a) is eliminated by using a filter;
  • FIG. 3 shows a photograph of a Western blot analysis of the expressed AtOEP7:GFP, wherein T, S, and M indicate the total protein extract, the soluble fraction, and the membrane fraction, respectively.
  • FIG 4 shows schematic diagrams of the reporter proteins targeting to the subcellular organelles by endosomal trafficking, which are classified as Group II proteins in the present invention.
  • Figure 5 shows fluorescence images showing the expression of the Group II reporter proteins in the cell, wherein
  • (b) shows the fluorescence image of sialtransferase (ST) translocated in the Golgi apparatus, wherein the red fluorescence signal is the auto-fluorescence signal of chloroplast; (c) shows that the reporter protein BiP:RFP is localized in the lumen of the endoplasmic reticulum;
  • Figure 7 shows fluorescent images showing the expression of the Group III reporter proteins, wherein
  • FIG. 8 shows photographs representing the inhibitory effect of wortmannin on the intracellular trafficking of a Group I reporter protein RbcS: GFP.
  • Wortmannin is known to be an inhibitor of PI(3)P and PI(4)R
  • Figure 9 shows photographs representing the effect of a chemical on the intracellular trafficking of a Group II reporter protein, wherein (a) and (b) show that the green fluorescence signal of 500:GFP:KKXX is observed as numerous networks in the control protoplast; and
  • Figure 10 shows photographs showing the change induced by a specific inhibitor upon co-expression of two reporter proteins, wherein
  • Figure 11 shows photographs that visualize the effects of brefeldin A disrupting the subcellular organelles, wherein (a) shows that BiP:RFP is distributed along the structure of the endoplasmic reticulum in the control protoplast;
  • Figure 13 shows photographs showing the variation in the intensity of the fluorescent signal induced by an expression inhibitor, wherein
  • the present invention provides a method for detecting specific characteristics related to trafficking and localization of a selected protein in a cell, which method comprises:
  • step (c) transforming the cell with at least one recombinant plasmid prepared in step (b);
  • the present invention provides a method for screening chemicals that affect specific characteristics related to trafficking and localization of a selected protein in a cell, which method comprises:
  • step (c) transforming the cell with at least one recombinant plasmid prepared in step (b);
  • the present invention provides recombinant genes encoding the reporter proteins that are used in the above methods to visualize the trafficking of the reporter proteins and their distributions in subcellular organelles.
  • the recombinant gene comprises a gene encoding a signal protein which includes a trafficking signal targeting to a specific subcellular organelle and a gene encoding a fluorescent protein linked thereto.
  • the signal protein is selected from the proteins that have trafficking signals targeting to nucleus, mitochondria, chloroplast, peroxisome, plasma membrane, endoplasmic reticulum, Golgi apparatus, storage vacuole, lytic vacuole, and prevacuolar compartment, and also those proteins targeting to 3 classes of phospolipids.
  • Examples of the signal proteins include NLS (nuclear localization sequence), AtOEP7, Cab (chlorophyll a/b binding protein), SKL (peroxisome targeting motif), RbcS (rubisco small subunit), RA (rubisco activase), Fl- H + -ATPase, H + -ATPase, BiP (chaperone binding protein), ST (Sialyltransf erase).
  • Chi chitinase
  • clone 491, clone 500 AtVTIla
  • SPO sporamin
  • EBD AtPH
  • FAPP PH
  • the present invention provides methods for preparing transformed cells that can express reporter proteins, each comprising one of the signal proteins described above and a fluorescent protein label linked thereto.
  • the present invention also provides a systematic method for selectively detecting the intracellular processes using the transformed cells and a systematic method for selectively screening drug candidates based on this detection method.
  • the methods of the present invention include a step of selecting a specific protein that has a property of translocating to a specific subcellular location, a step of synthesizing the whole gene of the selected signal protein or a portion thereof encoding the trafficking signal of the selected protein, a step of synthesizing a gene encoding a fluorescent protein that can be linked to the signal protein to fluorescently visualize the subcellular localization, and a step of constructing a recombinant gene comprising a gene encoding the signal protein and a gene encoding the fluorescent protein linked thereto.
  • the function of the signal protein can change depending on the way that the fluorescent protein is linked to the signal protein.
  • the present invention therefore provides compositions of the reporter proteins whose signal proteins can correctly direct the trafficking, and also construction methods thereof.
  • the present invention also provides a procedure for constructing recombinant plasmids that can be used to express the recombinant genes in a cell.
  • the recombinant plasmid can be constructed by ligating a recombinant gene into a vector containing a promoter, a terminator, and other necessary factors.
  • methods for transforming a cell by introducing the recombinant plasmid include, but are not limited to, chemical- mediated methods using PEG (polyethylene glycole), potassium phosphate, or DEAE- dextran, cationic lipid-mediated lipofection, microinjection, electroporation, and electrofusion.
  • one type of the recombinant plasmid could be introduced, or else two or more types of the recombinant plasmids can be introduced to express two or more reporter proteins simultaneously.
  • the conditions need to be optimized to efficiently express the reporter protein comprising the signal protein and the fluorescent protein in the transformed cell.
  • the persons skilled in the art can select appropriate conditions depending on the signal protein and the fluorescent protein used. Detailed structures of the recombinant plasmids are explained in the examples of the present invention to present the amino acid sequences of the reporter proteins or corresponding nucleic acid sequences.
  • the signal proteins were classified into three classes based on the protein trafficking mechanisms, in order to show that the methods of the present invention for constructing the reporter proteins can be commonly used for various mechanisms of protein trafficking to different subcellular organelles.
  • Signal proteins targeting to nucleus, chloroplast, mitochondria, etc. correspond to the case that a specific portion of the signal protein directly acts as a recognition signal to direct the intracellular trafficking.
  • These signal proteins are classified as Group ⁇ for convenience in the specification of the present invention (see Figure 1).
  • Signal proteins targeting to endoplasmic reticulum, Golgi apparatus, lytic vacuole, storage vacuole, plasma membrane, etc. correspond to the case of the endosomal trafficking in which a specific portion of the signal protein acts as a signal to be captured by endoplasmic reticulum so that the signal protein is translocated as enclosed in endoplasmic reticulum.
  • These signal proteins are classified as Group II for convenience in the specification of the present invention (see Figure 4). In the present invention, detailed methods are provided for visualizing the trafficking processes and the cellular distributions of these classes of the signal proteins.
  • signal proteins related to intracellular signal transduction via specific binding to phospolipids are selected in the present invention, and methods are provided for observing the subcellular organelles that contain specific phospholipid. These signal proteins are classified as Group III for convenience in the specifications of this invention (see Figure 6).
  • the present invention also provides a method for visualizing the localization of two or more proteins simultaneously by using two or more fluorescent proteins with different colors.
  • green fluorescent protein (GFP, Davis and Niestra, 1998) and red fluorescent protein (RFP) are used for constructing the reporter proteins to visualize their cellular localization.
  • RFP red fluorescent protein
  • the reporter proteins of the present invention can be constructed by using fluorescent proteins other than GFP and RFP.
  • the expression and trafficking processes of the reporter protein can be visualized in details for each stage of the processes by continuously monitoring the images of the fluorescence emitted by the reporter protein expressed in the transformed cell, using a fluorescence microscope at a specific wavelength.
  • a selective drug screening system for identifying chemicals inhibiting or enhancing the intracellular trafficking of the selected protein is established using this detection method. More particularly, it is demonstrated that chemicals affecting the intracellular trafficking can be identified by treating the transformed cell with a chemical before, after, or at the same time as the expression of the reporter protein, and then identifying the effect of the chemical by comparing the cellular distribution of the reporter protein in the transformed cell treated with the chemical with that in the control transformed cell which is not treated with the chemical.
  • the same method can be used to screen chemicals inhibiting or enhancing the transcription or the translation of proteins because the level of the protein expression can be examined from decrease or increase in the intensity of the fluorescence signal from the reporter protein. This is also demonstrated in the examples of the present invention.
  • Morphological changes induced by a chemical such as modification, damage, or destruction of the subcellular organelles can be detected by observing the distribution or pattern of the fluorescence signal from the reporter protein. Therefore, it is also possible to screen cytotoxic chemicals that cause alteration of the subcellular organelles. This is also demonstrated in the examples of the present invention.
  • inhibitors such as bafilomycin Al, wortmannin, and brefeldin A are examined to confirm that inhibition of the intracellular trafficking and expression processes and also cytotoxicity causing morphological changes of the subcellular organelles can be detected practically. This is described in detail in the examples of the present invention. As described in the examples, the signal proteins included in Group I, II, and III are examined to check the difference arising from the protein trafficking mechanisms.
  • Example 1 Construction of recombinant plasmids for expression of Group I proteins targeting to the organelles across the membrane.
  • the coding region for the transit peptide of Fl-H + -ATPase was amplified by polymerase chain reaction (PCR) from a ⁇ ZAPII cDNA library using two specific primers (5'-CTTTAATCAATGGCAATG (SEQ ID NO: 1) and 5'- CCATGGCCTGAACTGCTCTAAGCTT (SEQ ID NO: 2)) and ligated in-frame to the 5' end of the coding region of the green fluorescent protein to generate a recombinant gene for Fl- H + -ATPase:RFP (Niwa et al, 1999).
  • PCR polymerase chain reaction
  • the recombinant gene was subcloned into pUC under the control of the 35S promoter to construct a recombinant plasmid for ATPase:RFP. The same method was used for construction of other recombinant plasmids.
  • Rubisco ribulose bisphosphate carboxylase
  • the coding region for the transit peptide of the small subunit of the Rubisco complex was PCR amplified from a ⁇ ZAPII cDNA library using two specific primers (5'- CCTCAGTCACACAAAGAG (SEQ ID NO: 3) and 5'-
  • the coding region of the chloroplast a/b binding protein was PCR amplified from a ⁇ ZAPII cDNA library using two specific primers (5'-TAGAGAGAAACGATGGCG (SEQ ID NO: 5) and 5'-GGATCCCGTTTGGGAGTGGAACTCC (SEQ ID NO: 6)) and used to construct a recombinant plasmid for Cab: GFP .
  • the coding regions of the transit peptide of rubisco activase was PCR amplified from a ⁇ ZAPII cDNA library using two specific primers (5'- TCTAGAATGGCCGCCGCAGTTTCC (SEQ ID NO: 7) and 5'- GGATCCATCTGTCTCCATCGGTTTG (SEQ ID NO: 8)) and ligated to the 5' end of the coding regions of GFP and RFP to construct recombinant plasmids for RA:GFP and RA:RFP, respectively.
  • the coding region of the Arabidopsis outer envelope membrane protein, AtOEP7 a homolog of OEP14 of pea was PCR amplified from a Arabidopsis genomic DNA using two specific primers (OEP7-F: 5'-GACGACGACGCAGCGATG (SEQ ID NO: 9) and OEP7-R: 5'-GGATCCCCAAACCCTCTTTGGATGT (SEQ ID NO: 10)) which were designed to remove the natural termination codon, and subsequently ligated to the 5' end of the coding regions of GFP and RFP to construct recombinant plasmids for AtOEP7:GFP and AtOEP:RFP, respectively.
  • a recombinant plasmid for the nuclear localization signal (NLS), NLS:GFP, was constructed as described previously (Pih et al., 2000).
  • a recombinant plasmid for NLS:RFP was constructed by replacing the GFP coding region with the RFP coding region in the recombinant gene for NLS:GFP.
  • SKL which includes the proxisome targeting motif SKL (serine, lysine, leucine)
  • SKL proxisome targeting motif SKL (serine, lysine, leucine)
  • 326GFP Daavis and Niestra, 1998) using two specific primers (5'- CCGTATGTTACATCACC (SEQ ID NO: 11) and 5'-
  • the pelleted protoplasts were resuspended in 5 to 10 ml of the W5 solution (154 mM NaCl, 125 mM CaCl 2 , 5 mM KCI, 5 mM glucose, 1.5 mM Mes-KOH, pH 5.6), overlaid on top of 20 ml of 21% sucrose, and centrifuged for 10 min. at 78xg.
  • the intact protoplasts at the interface were transferred to a new tube containing 20 ml of the W5 solution.
  • the protoplasts were pelleted again by centrifugation at 55xg for 5 min and resuspended in 20 ml of the W5 solution.
  • the protoplasts were incubated on ice for 30 minutes.
  • Recombinant plasmids were purified using Qiagen columns (Valencia, CA) according to the manufacture's protocol. To transform the protoplasts with the DNA, the protoplasts were pelleted again by centrifuge at 46xg for 5 min and resuspended in the
  • MaMg solution 400 mM Mannitol, 15 mM MgCl 2 , 5 mM Mes-KOH, pH 5.6) at a density of
  • Plasmid DNA (about 20-50 ⁇ g at a concentration of 2 ⁇ g/ ⁇ l) was added to 300 ⁇ l of the protoplast suspension, and subsequently 325 ⁇ l of the PEG solution (400 mM Mannitol, 100 mM
  • the recombinant plasmids constructed in Example 1 were used to transform the protoplasts according to the method described in Example 2.
  • the expression of the reporter proteins after the transformation was monitored as a function of time by capturing images using a fluorescence microscope (Axioplan fluorescence microscope, Zeiss, Germany) equipped with a cooled charge-coupled device (CCD) camera.
  • the filter sets used were XF116 (exciter: 474AF20, dichroic: 500DRLP, emitter: 510AF23), XF33/E (exciter: 535DF35, dichroic: 570DRLP; emitter, 605DF50), and XF137 (exciter, 540AF30; dichroic, 570DRLP, emitter: 585ALP) (Omega, Inc, Brattleboro, VT) for GFP, RFP, and auto- fluorescence of chlorophyll, respectively. Data were then processed using Adobe (Mountain View, CA) Photoshop software, and the images were rendered in pseudo-color.
  • the green fluorescence of the reporter protein AtOEP7:GFP was observed at the outer envelop membrane of the chloroplast.
  • the red fluorescence in Figure 2a is the auto- fluorescence of chloroplasts.
  • Figure 2b shows the image obtained by eliminating this auto- fluorescence by using a filter. This result indicates that the fusion protein comprising the signal protein with the chloroplast envelope targeting signal and the fluorescent protein label was correctly targeted to the chloroplast envelope membrane.
  • RbcS: GFP, Cab: GFP, and RA:GFP are presented in Figure 2(c), 2(d), and 2(e), respectively.
  • RbcS:GFP was targeted to the stroma of chloroplast, and Cab:GFP and RA:GFP also emitted the fluorescence in the chloroplast.
  • Example 4 Confirmation of the chloroplast envelope targeting of AtOEP7:GFP by Western blot analysis.
  • the recombinant plasmid for AtOEP7:GFP was constructed according to the method in Example 1. This recombinant plasmid was used to transform protoplasts according to the method in Example 2, and the transformed protoplasts were incubated for 24 hrs at 22°C.
  • the total protein extract was prepared as follows.
  • the result shows that the expressed signal protein was transported to the chloroplast envelope membrane and not present in the cytosol.
  • This result indicates that localization of proteins, which is conventionally determined by Western blot analysis, can be identified by the method provided by the present invention.
  • Example 5 Construction of recombinant plasmids for expression of Group II proteins that are transported to subcellular organelles by endosomal trafficking.
  • H + -ATPase (Arabidopsis AHA2) was amplified with two specific primers (5'-GAGATGTCGAGTCTCGAA (SEQ ID NO: 13) and 5'- CTCGAGCACAGTGTAGTGACTGG (SEQ ID NO: 14)) and ligated to the 5' end of the GFP coding sequence.
  • the ligated recombinant gene was subcloned into the pUC vector under the control of the 35S promoter to construct a recombinant plasmid for H + - ATPase:GFP. The same procedure was applied in the following examples.
  • the coding sequence of the chaperone binding protein (BiP) (access number D82817) was amplified from an Arabidodsis cDNA library using two specific primers, BIP5 (5'-TACGCAAAAGTTTCCGAT-3' (SEQ ID NO: 15)) and BIP3 (5'- CTAGAGCTCATCGTGAGA-3' (SEQ ID NO: 16)).
  • BIP5 5'-TACGCAAAAGTTTCCGAT-3' (SEQ ID NO: 15)
  • BIP3 5'- CTAGAGCTCATCGTGAGA-3' (SEQ ID NO: 16)
  • the amino terminal region (44 amino acids) and the carboxyl terminal region (80 amino acids) of this gene were ligated to the amino terminus and the carboxyl terminus of GFP or RFP, respectively, to construct recombinant plasmids for BiP:GFP and BiP:RFP.
  • the sialtransferase (ST) cDNA was amplified from a ⁇ ZAPII cDNA library using two specific primers (5'-ATGATTCATACCAACTTGAAG (SEQ ID NO: 17) and 5'- GGATCCACAACGAATGTTCCGGAA (SEQ ID NO: 18)). GFP or RFP was ligated in- frame to the carboxyl terminus of ST to construct ST: GFP or ST:RFP.
  • a recombinant plasmid for 500 was constructed by inserting the GFP coding region without the termination codon into the EcoRI site of clone 500 (Kim et al., 2001).
  • plasmid for 526 GFP was constructed by inserting the coding region of GFP into the EcoRI site of clone 526.
  • recombinant plasmids for 491:GFP and 491:RFP were constructed by inserting clone 491 into the 5' end of the coding regions of GFP and RFP without the termination codon, respectively.
  • a recombinant plasmid for 500:GFP:KKXX was generated as follows: The GFP coding region without the termination codon was inserted into the EcoRI site of clone 500 (Jiang and Rogers, 1998) and KKXX was then added to the C-terminus of 500:GFP by PCR amplification using two specific primers (5'-GGATCCTCTAGAGGATCGATCCGG (S ⁇ Q ID NO: 19) and 5'-
  • AtVTIla a homolog of Arabidopsis t-SNAR ⁇ which is transported from the tr ⁇ s-Golgi network to the storage prevacuole (Zheng et al., 1999), a recombinant plasmid for RFP:AtNTTla was constructed by ligating the coding region of AtNTIla to the C-terminus of the RFP coding region.
  • a recombinant plasmid for AtVTI GFP was constructed by ligating the coding region of GFP to the C-terminus of the coding region of AtNTIla.
  • a recombinant plasmid for SPO:GFP was constructed by ligating GFP to the carboxyl terminus of the sporamin B gene.
  • Schematic diagrams of the reporter proteins expressed from the recombinant plasmids constructed as above are shown in Figure 4.
  • Example 6 Observation of the expression and localization of Group II reporter proteins.
  • Recombinant plasmids for H + -ATPase:GFP, ST:GFP, BiP:GFP, 526:GFP, Chi- n:RFP:Chi-c, and 500:GFP:KKXX were constructed as described in Example 5 and used to transform the protoplasts by the method of Example 2. Expression of the reporter proteins was monitored as a function of time using a fluorescence microscope as explained in Example 3. A part of the results is given in the following.
  • Chi-n:RFP in which the carboxyl region of chitinase was not ligated, was not targeted to the storage vacuole, but it was present as speckles in the endoplasmic reticulum ( Figure 5(g)). Fluorescence of SPO:GFP was distributed uniformly throughout the lytic vacuole ( Figure 5(h)).
  • Example 7 Construction of recombinant plasmids to express Group III proteins that are specific to phospholipids.
  • the C-terminal coding region (amino acid residue 1257 to 1411) of human early endosome antigen 1 (EEA1) was PCR amplified with two primers 5'-GAATTCGTGGCAATCTAGTCAACGG-3' (SEQ ID NO: 21) and 5'-CTAATGTTAGTGTAATATTAC-3' (SEQ ID NO: 22), and ligated to the C- terminus of the GFP coding sequence without the termination codon.
  • This recombinant DNA was inserted to a pUC vector under the control of the 35S promoter to construct a recombinant plasmid. The same cloning procedure was applied in the examples hereafter.
  • a recombinant plasmid for a EBD derivative, GFP.EBDC1358S was prepared using a primer directing replacement of the amino acid residue 1358 to serine.
  • a recombinant plasmid for the fusion protein of Arabidopsis Pleckstrin homology (PH) domain, GFPAtPH was constructed by PCR amplification using two primers 5'- CCCGGGAAATGGAGAGTATGTGGCGA-3' (SEQ ID NO: 23) and 5'- TAATCACCGCCTGTGATCATA-3' (SEQ ID NO: 24).
  • a recombinant plasmid for the fusion protein of FAPP including the PH domain, GFP:FAPP was constructed by PCR amplification using two primers 5'-CTCGAGATGGAGGGGGTTCTGTACAAG-3' (SEQ ID NO: 25) and 5'-TCACGCTTTGGAGCTCCCAAGGGC-3' (SEQ ID NO: 26).
  • a recombinant plasmid for PH:GFP was constructed by the method of Kost B et al. (1998).
  • Example 8 Observation of the expression and localization of Group III reporter proteins.
  • Recombinant plasmids for GFP:EBD, GFP:AtPH, GFP:FAPP, and GFP:PH were constructed as described in Example 7 and used to transform the protoplasts by the method of Example 2. Expression of the reporter proteins was monitored as a function of time using a fluorescence microscope as explained in Example 3. A part of the results is given in the following.
  • PI(4)P phosphatidylinositol 4-phosphate
  • PI(4,5)P2 phosphatidylinositol 4,5-diphosphate
  • Example 9 The effect of wortmannin on the intracellular trafficking of RbcS: GFP.
  • a recombinant plasmid for RbcS: GFP was constructed as described in Example 1.
  • Example 2 Isolation of the recombinant plasmid, preparation of protoplasts, and transformation of protoplasts were performed as in Example 2.
  • the protoplast suspension was treated with wortmannin at a concentration of 5 ⁇ g/ml. Then the protoplasts were transformed and incubated in the dark. Expression of the fusion protein was observed as in Example 3.
  • Wortmannin is known as a specific inhibitor of phosphatidyl 3-phosphate (PI(3)P) and phosphatidylinositol 4-phosphate (PI(4)P) (Ui et al., 1995). Localization of the green fluorescence in the wortmannin-treated protoplast was compared with that of the control protoplast that was not treated with wortmannin. As shown in Figure 8, in contrast to the control protoplast in which the green fluorescence of RbcS: GFP was targeted to the chloroplast as expected ( Figure 8(a)), the green fluorescence was not translocated to the chloroplast in the presence of wortmannin, but observed as either speckles or aggregates ( Figure 8(b) and 8(c)). This result indicates that wortmannin inhibits trafficking of the chloroplast-targeting protein from the cytosol to the chloroplast.
  • PI(3)P phosphatidyl 3-phosphate
  • PI(4)P phosphatidylinositol 4-phosphate
  • Example 10 The effect of bafilomycin Al (BafAl), known as an inhibitor of the vacuolar type H + -ATPase, on retrograde trafficking of 500:GFP:KKXX.
  • the recombinant plasmid for 500:GFP:KKXX was constructed as described in Example 5. Isolation of the recombinant plasmid, preparation of protoplasts, and transformation of protoplasts were performed as in Example 2. The protoplast suspension was treated with bafilomycin Al at a concentration of 5 ⁇ g/ml. Then the protoplasts were transformed and incubated in the dark. Expression of the fusion protein was observed as in Example 3.
  • BFA Brefeldin A
  • Arfs ADP-ribosylation factors
  • Example 12 The effect of brefeldin A (BFA) on the biogenesis and structure conservation of the endoplasmic reticulum.
  • a recombinant plasmid for BiP:RFP was constructed as described in Example 5.
  • Example 2 Isolation of the recombinant plasmid, preparation of protoplasts, and transformation of protoplasts were performed as in Example 2.
  • the protoplast suspension was treated with brefeldin A at a concentration of 5 ⁇ g/ml. Then the protoplasts were transformed and incubated in the dark. Expression of the fusion protein was observed as in Example 3.
  • Examplel3 Inhibition of trafficking of proteins that are specific to phospholipids.
  • the transformed protoplasts were treated with wortmannin at a concentration of 1.0 ⁇ g/ml or with 2-(4-morpholinyl)-8- phenyl-4H-l-benzopyran-4-on, a specific inhibitor of phosphatidylinositol 3-kinase, at a concentration of 10 ⁇ g/ml (LY294002, Nlahos et al., 1994) and incubated at 22°C. Fluorescence images were monitored at various time points.
  • Example 14 Change in the expression level induced by cycloheximide, an inhibitor of the protein expression.
  • Example 2 Isolation of the recombinant plasmid, preparation of protoplasts, and transformation of protoplasts were performed as in Example 2.
  • the protoplast suspension was treated with cycloheximide a concentration of 5 ⁇ g/ml. Then the protoplasts were transformed and incubated at 22°C in the dark. Expression of the fusion protein was observed as in Example 3.
  • a new Dynamin-like protein, ADL6 is involved in trafficking from the tr ns-Golgi network to the central vacuole in Arabidopsis. Plant Cell (in press)
  • a GFP-mouse talin fusion protein labels plant actin filaments in vivo and visualizes the actin cytoskeleton in growing pollen tubes. Plant J. 16, 393-401.
  • the plant vesicle-associated SNARE AtNTIla likely mediates vesicle transport from the trans-Golgi network to the prevacuolar compartment. Mol. Biol. Cell 10, 2251-2264.

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Abstract

L'invention concerne un procédé permettant de détecter de manière sélective des caractéristiques spécifiques se rapportant à la circulation intracellulaire et à la localisation infracellulaire d'une protéine choisie par observation d'une image par fluorescence. Elle concerne également un procédé de criblage de manière sélective de produits chimiques qui affectent de telles caractéristiques spécifiques de la protéine dans une cellule.
PCT/KR2002/001345 2001-07-16 2002-07-16 Systeme de surveillance d'un processus intracellulaire et criblage in vivo de medicaments WO2003008536A2 (fr)

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EP1298435A3 (fr) * 2001-09-28 2004-01-14 Genoptera, LLC Essai pour composés affectant la voie sécrétoire cellulaire des invertébrés
WO2006040340A3 (fr) * 2004-10-15 2006-08-31 Dsm Ip Assets Bv Procede pour la production d'un compose dans une cellule eucaryote
FR2920878A1 (fr) * 2007-09-10 2009-03-13 Innovative Concepts In Drug De Procede de toxicologie predictive ou de test d'efficacite par mesure de mobilite d'organites
CN108676075A (zh) * 2018-06-01 2018-10-19 山西省农业科学院果树研究所 一种引导蛋白分子进入植物细胞核的方法
WO2021088944A1 (fr) * 2019-11-07 2021-05-14 中国科学院遗传与发育生物学研究所 Utilisation de chaperon moléculaire groel/es d'escherichia coli pour aider à la synthèse de rubisco de plante
CN117230109A (zh) * 2023-11-16 2023-12-15 三亚中国农业科学院国家南繁研究院 代替荧光蛋白的可视化质粒及其相关应用
CN119101709A (zh) * 2024-09-10 2024-12-10 深圳市第三人民医院(深圳市肝病研究所) 人源trem2蛋白的双重荧光标记活细胞样品及其制备方法和应用

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KR100863283B1 (ko) * 2002-10-11 2008-10-15 아람 바이오시스템 주식회사 세포내 과정의 관측 방법 및 그를 위해 사용되는 재조합유전자
CN109136321A (zh) * 2018-08-29 2019-01-04 北京艾普希隆生物科技有限公司 一种植物亚细胞定位试剂盒及其应用
KR102288367B1 (ko) * 2018-11-15 2021-08-11 주식회사 바이오앱 식물체에서 바이러스-유사 입자를 발현하는 재조합 벡터 및 이를 이용한 써코바이러스-유사 입자를 포함하는 백신 조성물의 제조방법
CN114317593A (zh) * 2021-12-31 2022-04-12 北京林业大学 一种超分辨显微技术观察植物原生质体膜蛋白动态的方法

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US6020192A (en) * 1996-01-18 2000-02-01 University Of Florida Humanized green fluorescent protein genes and methods

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1298435A3 (fr) * 2001-09-28 2004-01-14 Genoptera, LLC Essai pour composés affectant la voie sécrétoire cellulaire des invertébrés
US8129143B2 (en) 2004-10-15 2012-03-06 Dsm Ip Assets B.V. Method for production of a compound in a eukaryotic cell
WO2006040340A3 (fr) * 2004-10-15 2006-08-31 Dsm Ip Assets Bv Procede pour la production d'un compose dans une cellule eucaryote
JP2008516592A (ja) * 2004-10-15 2008-05-22 ディーエスエム アイピー アセッツ ビー．ブイ． 真核細胞における化合物の生成方法
US8497089B2 (en) 2007-09-10 2013-07-30 Innovative Concepts In Drug Development (Icdd) Method to predict toxicity using the analysis of dynamic organelle behaviour
WO2009034094A1 (fr) * 2007-09-10 2009-03-19 Innovative Concepts In Drug Development (Icdd) Procédé de prévision de la toxicité utilisant l'analyse du comportement dynamique des organites
FR2920878A1 (fr) * 2007-09-10 2009-03-13 Innovative Concepts In Drug De Procede de toxicologie predictive ou de test d'efficacite par mesure de mobilite d'organites
CN108676075A (zh) * 2018-06-01 2018-10-19 山西省农业科学院果树研究所 一种引导蛋白分子进入植物细胞核的方法
CN108676075B (zh) * 2018-06-01 2022-04-01 山西省农业科学院果树研究所 一种引导蛋白分子进入植物细胞核的方法
WO2021088944A1 (fr) * 2019-11-07 2021-05-14 中国科学院遗传与发育生物学研究所 Utilisation de chaperon moléculaire groel/es d'escherichia coli pour aider à la synthèse de rubisco de plante
CN117230109A (zh) * 2023-11-16 2023-12-15 三亚中国农业科学院国家南繁研究院 代替荧光蛋白的可视化质粒及其相关应用
CN117230109B (zh) * 2023-11-16 2024-03-19 三亚中国农业科学院国家南繁研究院 代替荧光蛋白的可视化质粒及其相关应用
CN119101709A (zh) * 2024-09-10 2024-12-10 深圳市第三人民医院(深圳市肝病研究所) 人源trem2蛋白的双重荧光标记活细胞样品及其制备方法和应用

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