WO2004019967A1 - Method for isolating subpopulations of proteins that engage in protein-protein interactions - Google Patents
Method for isolating subpopulations of proteins that engage in protein-protein interactions Download PDFInfo
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- WO2004019967A1 WO2004019967A1 PCT/US2002/025845 US0225845W WO2004019967A1 WO 2004019967 A1 WO2004019967 A1 WO 2004019967A1 US 0225845 W US0225845 W US 0225845W WO 2004019967 A1 WO2004019967 A1 WO 2004019967A1
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- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B30/00—Methods of screening libraries
- C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
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- 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
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- 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
Definitions
- the invention relates to the study of protein-protein interactions and is expected to be useful in the fields of biochemical signal transduction, proteomics, drug discovery, toxicology, and diagnostics.
- Protein-protein interactions underlie a vast number of physiological processes.
- Cellular processes such as neuronal signaling, cell development, growth, and replication all depend on a complex network of protein-protein and protein-small molecule interactions in the cell. These interactions may be categorized as constitutive interactions, such as between subunits of hemoglobin, and signal-dependent interactions, such as those between the subunits of cAMP-dependent protein kinase or the subunits of GTP-binding proteins.
- the complexity of the task of investigating these interactions is evident from the potential number of protein interactions: comprehensively screening binary interactions among 15,000 proteins would require testing over 2 *10 8 pairwise combinations of proteins. This complexity means that conventional biochemical methods are of limited use. Despite intensive research, there is still no satisfactory method for systematically studying protein interactions in mammalian cells or other complex mixtures of proteins.
- TAP tandem affinity purification
- SlBS UTE SHEET (RULE 26) were rendered "high-throughput" by a brute force approach, which involved individually processing over 1,700 genes and over 1,000 individual yeast expression clones in the former case and 725 genes in the latter.
- TAP tagging method also exhibits a bias against proteins below 15kDa, and both TAP and epitope tags may interfere with normal protein- protein interactions.
- the invention provides a method for screening of both constitutive and signal-mediated protein-protein interactions.
- the method of the invention has several advantages:
- the method uses an activated solid support to isolate proteins that are non- covalently bound to other proteins.
- the support is preferably a gel, and is more preferably agarose.
- the support is activated by the presence of chemically-reactive functional groups that are capable of covalently binding proteins. Cyanogen bromide-activated Sepharose(TM) is a preferred support. Because the interacting proteins are subject to experimental environmental manipulation, mass spectrometric identification of the proteins can yield information on specific classes of interacting proteins, such as calcium-dependent or substrate-dependent protein-protein interactions. This permits the selection and isolation of a subpopulation of proteins from a complex mixture on the basis of specified interaction criteria.
- the method enables the simultaneous screening of an entire proteome, unlike two-hybrid systems or phage display which can only detect proteins binding to a single bait protein at a time. Since only naturally-occurring interactions of proteins in their native state are observed, this method will have wide applicability to studies of protein interactions in tissue samples and autopsy specimens, for screening for perturbations of protein-protein interactions by signaling molecules, pharmacological agents or toxins, and screening for differences between cancerous and untransformed cells.
- the invention provides a method of isolating, from a mixture of proteins, a subpopulation consisting essentially of proteins that engage in protein-protein interactions.
- the method comprises the steps of (a) contacting the protein mixture with a chemically reactive support, under conditions that permit both covalent binding of proteins to the support and protein-protein interactions; (b) permitting proteins in the mixture to become covalently bound to the support; (c) separating the support from any proteins not bound to it; (d) subjecting the support to conditions that disrupt protein-protein interactions; and finally (e) separating the support from any proteins not bound to it.
- the proteins released in step (e) are those proteins that non-covalently bound other proteins under the conditions of step (a).
- the chemically reactive support may contain any chemically reactive functional group capable of covalently binding proteins in an aqueous environment.
- Preferred chemically reactive moieties include but are not limited to cyanate, isocyanate, isothiocyanate, activated carboxyl, activated sulfonyl, aldehyde, epoxide, and thiol groups. Particularly preferred is the cyanate group.
- the support may be any matrix that is physically separable from the reaction mixtures.
- the support is preferably in the form of particles or beads.
- the support comprises an optionally cross-linked polymer or gel.
- Preferred support materials include but are not limited to polystyrene, agar, agarose, polyacrylamide, dextran, hydroxylated vinyl polymers, and carboxylated vinyl polymers.
- a particularly preferred support comprises agarose, for example the varieties of cross-linked agarose sold under the trade name Sepharose(TM).
- the methods of this invention are also useful in the analysis of protein interactions with protein microarrays.
- an entire proteome is applied to a microarray of immobilized proteins to investigate protein-protein interactions.
- any specifically-binding proteins of interest are in competition with an enormous excess of proteins that do not participate in specific protein interactions. These excess proteins may be adsorbed nonspecifically onto the microarray and/or compete for binding sites by virtue of their greater concentration in the mixture.
- the relatively large mass of protein requires a proportionally large volume of solubilizing buffer, which reduces the concentration of proteins of interest. Through concentration effects, fluorescence quenching, competition, and dilution, the presence of a large quantity of irrelevant proteins can greatly reduce the signal-to-noise ratio obtained from a protein microarray.
- the present invention reduces these problems by pre-selecting a subpopulation of proteins on the basis of their ability to interact with other protein targets.
- This subpopulation contains precisely those proteins that are likely to bind specifically to a protein microarray.
- the method of the invention will reduce the total number of distinct proteins in a proteome by at least 75% prior to application to a protein microarray.
- the proteins that are eliminated are those that do not participate in specific protein-protein interactions, including many that have the potential to be non-specifically adsorbed or trapped on the array.
- the retained proteins may be labeled if desired (e.g., with biotin or an appropriate fluorescent dye) and applied to a protein microarray in the same manner as is
- SUBSTIT ⁇ TE SHEET (RULE 26) currently done in existing applications. In so doing, the potential noise on a protein microarray from the unwanted proteins is reduced considerably, resulting in a substantial improvement in the quality of the results obtainable from protein microarrays.
- Another application of the method to microarrays results from the ability of the method to easily isolate protein subpopulations with specific, desired biochemical properties. For example, the practitioner may use the method to select for proteins whose interactions depend on the presence of calcium, cAMP, a specific DNA sequence, or a pharmacological agent such as rapamycin. By manipulating the wash conditions, proteins with relatively low or relatively high affinity may be selected for. When the method of the invention is employed to preselect for proteins of interest, the microarray is more likely to successfully identify the proteins involved in an interaction. The method permits researchers to perform tests that would otherwise require construction of specialized microarrays or other expensive or indirect methods to achieve similar results.
- the method of the invention provides an improvement which consists of isolating, from the mixture of proteins, a subpopulation consisting essentially of proteins that engage in protein-protein interactions, before the subpopulation is subsequently contacted with the array.
- Figure 1 A shows the six possible outcomes of binding a bimolecular protein complex (AB) to Sepharose(TM) support particles.
- Figure 1 B illustrates the elution of non-covalently associated proteins from the Sepharose(TM) particles of Fig. 1 A.
- Figure 2 is a Western blot of an SDS-PAGE gel, comparing the calmodulin- binding proteins isolated using the CNBr-Sepharose(TM) method (left) to those isolated by calmodulin- affinity chromatography (right).
- Figure 3 Coomassie blue-stained 2-dimensional gel of rat brain extract subjected to enrichment of Ca2+ -dependent protein-protein interactions by the method of the invention. Fewer than 200 spots are visible on this gel, indicating a selected subpopulation of proteins. Of the 23 largest spots, the 12 indicated spots were identified by mass spectrometry.
- Figure 4 is a 2-dimensional gel of rat brain proteins released from CNBr-
- Sepharose with 8M urea Sepharose with 8M urea.
- Figure 5 shows the calmodulin spot in a 2-dimensional gel after selection for
- Figure 6 shows a potential application of the method in the investigation of learning-dependent changes in protein interactions.
- the panels are corresponding regions of 2-D polyacrylamide gels of proteins from hippocampal extracts of rats trained (left panel) and untrained (right panel) in a water maze. Two proteins (center of left panel) are candidates for learning-specific alterations in protein-protein interactions.
- CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-l-propane sulfonate
- EGTA Ethylene glycol bis-(2-aminoethyl eff ⁇ er)-N,N,N',N'- tetraacetic acid
- MARCKS Myristoylated Alanine-Rich C Kinase Substrate
- Non-physiologically relevant interactions are a significant potential problem in many studies of protein-protein interactions, including the two-hybrid system.
- nonspecific protein interactions will also occur between proteins that normally are not in contact with each other. For example, interactions between membrane and nuclear proteins, or between astrocyte and neuronal proteins could occur.
- SUBS1 ⁇ TUTE SHEET (RULE 26)
- the method could be used to study protein interactions that may occur after associative learning in the rat water maze task [24, 25].
- This experiment uses a trained group, which swims in a tank of water containing a concealed platform, and an untrained control group, which is allowed to swim in a tank with no platform. Interacting proteins from hippocampal extracts of each group would then be isolated separately using the CNBr-Sepharose(TM) method and analyzed on 2-dimensional polyacrylamide gels. Any differences in protein interactions produced by learning would be reflected as spots for which the intensity in the trained group differs from the intensity of the corresponding protein spot in the control groups.
- Figure 6 demonstrates the results from such an experiment, with two proteins possibly exhibiting a learning- specific increase in protein interactions (upper center in left panel). Once these proteins are identified by mass spectrometry or other means, their binding partners can be easily identified. This could provide a useful means of identifying new signaling pathways relevant to physiological processes.
- the calcium or pharmacological agent being tested would be added to the buffer at each step in sufficient concentration to ensure tight binding of all relevant proteins.
- the proteins would be eluted by washing the chromatography column with a buffer identical in all respects except that the pharmacological agent is omitted.
- the ionic strength and pH of the two buffers should be identical to avoid eluting any proteins by virtue of a change in pH or ion concentration. In this case, it is not strictly necessary to compare two separate samples, because the protein interactions of interest are those occurring in the test tube.
- SUBSTIT ⁇ TE SHEET (RULE 26) [0040]
- the data produced using this method will consist of raw information concerning proteins that may interact with some other, as yet unidentified protein or proteins. Although this is valuable information in itself, and reduces the problem space by several orders of magnitude, it is still necessary to validate the putative protein interactions. Once the target proteins are identified, their binding partners can be easily found using conventional techniques such as affinity chromatography [27] or two-hybrid analysis. To provide a complete understanding of the interaction, it also necessary to confirm that the putative interaction occurs using some alternative method. Confirmation of the observed change is, of course, also necessary in other screening methods such as DNA microarrays, phage display, or the two-hybrid system.
- This technique could also be modified by adding a cross-linking step after the initial wash, and substituting thiol-Sepharose(TM) for CNBr-Sepharose(TM). This would permit the pair of interacting proteins to be separated by cleavage of the disulfide bond linking the protein to Sepharose(TM), allowing the crosslinked protein pair to be separated and identified as a single unit.
- the new method has the advantage of screening the entire proteome simultaneously, unlike other methods which can only detect proteins binding to a single bait protein at a time.
- the method does not require cloning but isolates naturally-occurring interactions between proteins in their native, folded state that are properly post-translationally modified.
- the proteins are also accessible to chemical manipulation, permitting selection of a subpopulation of proteins from a complex mixture on the basis of specified interaction criteria.
- the method would be useful not only for studying protein-protein interactions, but also for identifying the site of action of low- molecular-weight compounds such as xenobiotics or pharmacological agents. Previously, determining whether a xenobiotic affected protein-protein interactions was a daunting task
- Sepharose(TM) particles are assumed to be interchangeable. Three additional outcomes are possible in which A and B are switched. Due to the relatively large size of the particle, outcomes in which the protein complex is bound to different particles (1, 3, 4, and 5 in Fig. 1 A) are eliminated because mechanical stress overcomes the chemical bond. Outcomes in which both partners are bound to the same particle (6) should be relatively rare so long as the density of activated groups on the particle is not too high. This leaves only outcome (2), in which one protein is covalently bound to the particle and the other is non-covalently associated with it.
- the protein mixture is reacted with cyanogen bromide-activated Sepharose(TM) in such a way that 50% of the proteins are covalently bound.
- the remaining proteins are either washed away or retained on the
- SUB5 m [RULE 21 Sepharose(TM) by interacting noncovalently with the covalently-bound proteins.
- the principal components would be proteins bound at a single site (outcome 2), in which one partner is covalently attached to a particle and its noncovalently attached interacting partner is retained by virtue of its affinity to the bound protein.
- the noncovalently attached protein is then eluted by washing in 8M urea (Fig. IB).
- the elution buffer can be modified to examine specific types of protein- protein interactions, such as substrate-dependent or calcium-dependent interactions.
- the eluted proteins are analyzed by an appropriate method, such as 2-D gel electrophoresis or capillary LC-MS [28, 29].
- Eluted proteins were concentrated, desalted, and separated by 1 -dimensional SDS-polyacrylamide gel electrophoresis, blotted onto nitrocellulose, and stained using antibody against calmodulin dependent kinase I, calmodulin dependent kinase II, MARCKS, or protein phosphatase 2A.
- the Western blot analysis ( Figure 2, left lanes) showed that all four calmodulin-binding proteins tested (CaM kinase I and II, MARCKS, and protein phosphatase 2A) were detectable with the method.
- the blank (“blk” lane) was from a sample in which brain extract was loaded onto Sepharose(TM) rendered inert by reacting with Tris- HC1.
- SUBSTIT ⁇ TE SHEET (RULE 26) is capable of enriching a subpopulation of proteins from a very complex mixture on the basis of a specific class of protein interactions.
- a sample of rat brain extract was bound to CNBr-Sepharose(TM) as before, the EGTA-eluted proteins were separated by 2- dimensional polyacrylamide gel electrophoresis.
- Fig. 3 shows a Coomassie-stained gel from this experiment. The total number of measurable spots (approx. 172) was smaller than the 300-400 spots visible when all interacting proteins were eluted with 8M urea (Fig. 4), and much smaller than the 1000-1200 spots routinely visible from unselected extract (not shown). Twenty-three of the more intense spots detected on the 2-D gel were subjected to digestion with trypsin, and the resulting peptides analyzed by LC-MS/MS.
- spot #1 The most abundant protein spot on the 2-dimensional gel (Spot #1) was identified as the calcium-binding protein calmodulin (Table 1 and Fig. 3). This spot is also detectable in crude extract, but is a relatively minor component (Fig. 5). Most of the remaining identifiable spots, including ATP synthase, mitochondrial ATPase inhibitor, and heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2), are also either known calcium- binding proteins or proteins that interact directly with calcium-binding proteins [30, 31, 32, 33, 34]. Peptides from S-100, another calcium-binding protein that undergoes numerous calcium-dependent protein interactions [35], were also detected. Although the M and pi were identical with S-100, because of the small number of observed peptides in the digestion, the mass spectrometric identification did not reach statistical significance.
- Table 1 summarizes the proteins identified by mass spectrometry. With the exception of hemoglobin, citrate synthase, and carbonic anhydrase, all the proteins identified were either known calcium-binding proteins or were proteins with well- characterized calcium-dependent interactions. For example, tropomyosin is associated with the well-known actin-troponin-myosin complex. Ca 2+ binding to troponin enables troponin to bind tropomyosin and shift it from myosin's binding sites on the actin proteins. Without
- troponin is no longer able to bind to tropomyosin, tropomyosin again blocks myosin's binding sites on the actin proteins.
- Tropomyosin also binds to the calcium- binding protein calcyclin [36].
- Rho GDP dissociation inhibitor strongly binds to the low-MW GTP-binding protein rho, which participates with the calcium binding protein cadherin in reorganization of actin cytoskeleton [37].
- Calponin is also a substrate of rho- kinase [38].
- Rho GDI-1 Binds Cadherin via Rho
- hemoglobin Three unexpected proteins, hemoglobin, carbonic anhydrase 2, and citrate synthase, were also detected. Although hemoglobin binding to other hemoglobin subunits depends on Fe 2+ and O 2 , it is not known to bind calcium; however, hemoglobin can bind to reticulocyte membranes in the presence of calcium [40], suggesting that it may be a partner
- SUBSTIT ⁇ TE SHEET (RULE 26) for a calcium-binding membrane-bound protein.
- citrate synthase and carbonic anhydrase can associate with as-yet uncharacterized proteins in the presence of calcium.
- the method described here should be useful for investigating protein-protein interactions in mammalian tissues. For example, it has been suggested that Alzheimer's disease and other neurodegenerative disorders are triggered by pathological protein-protein interactions [41, 42]. Similarly, cell signaling, synaptic plasticity, learning, and development are dependent on a complex network of protein-protein interactions. This method is expected to be useful in isolating macromolecular protein complexes as part of any program of proteomic screening to identify relevant protein-protein interactions for further study.
- Sepharose(TM) 4B (Pharmacia) was rehydrated and washed 3 times with water before use. CNBr was titrated with rat brain extract by incubating a fixed quantity of extract at room temperature with varying amounts of CNBr Sepharose(TM). After 1 hr, samples were centrifuged and the unbound protein was measured using a dye-binding assay [43] and the quantity of CNBr Sepharose(TM) to reduce the protein concentration by 50%> was calculated.
- Isolation of interacting proteins One rat brain was homogenized by sonication in 10 mM NaHCO 3 , pH 7.1 containing 5% CHAPS, 0.1 mM phenylmethylsulfonyl chloride, and 1 mM CaCl 2 , and centrifuged at 100,000g for 20 min. A quantity of rehydrated CNBr Sepharose(TM) sufficient to bind 50%> of the protein was added, and the sample was shaken at room temperature for 1 hr. Tris acetate was then added to 0.1 M to block unreacted CNBr and incubation was continued for another 30 min.
- SUBS ⁇ iTd ⁇ E SHEET (RULE 23) isoelectric focusing strip that had been rehydrated with the same solution, and subjected to flatbed 2-dimensional polyacrylamide gel electrophoresis (ExcelGel 12-14).
- Affinity chromatography Rat brain extract was incubated for 15 min at room temperature with 2 cm3 of calmodulin-Sepharose(TM) 4B in 50 mM NaHCO , pH 7.7 and 1 mM CaCl 2 . The mixture was transferred to a column and washed with 100 mM Tris-HCl containing 1 mM CaCl 2 until A 80 became undetectable. The calmodulin-binding proteins were eluted with 50 mM EGTA, desalted and concentrated in a Centricon-3 ultrafiltration device, separated by electrophoresis in a A-20% SDS polyacrylamide gel, and blotted onto nitrocellulose membranes.
- Mass spectrometrv Stained protein spots were excised from the 2- dimensional gel and digested with trypsin, using the in-gel method described by the Association of Biomolecular Resource Facilities [44]. Digestion with trypsin was carried out overnight at 37° C, and peptides were extracted from the gel into 5% formic acid:acetronitrile (1:1), and a second extraction into 5% formic acid:acetonitrile (5:95). The extracts were pooled, the volume reduced by vacuum centrifugation, and the final volume was brought up to 10 microliters with 0.1 %> TFA.
- Contaminating salts and particulates were removed by binding the peptides to a C ⁇ 8 -ZipTip (Millipore, MA), washing with 0.1%> TFA, and elution into 10 microliters of 0.1%> TFA: acetonitrile (1:1).
- the peptides from the tryptic digests were analyzed by tandem liquid chromatography/mass spectrometry (LC-MS/MS). Liquid chromatography was performed using a Michrom Magic HPLC system with a constant pressure splitter to reduce the flow rate through the column to 400 nl/min.
- Peptides were separated by reversed phase chromatography, using Vydac C ⁇ 8 , 5 micrometer particle, 300 angstrom pore packing. A column of approximately 5 cm was packed into a 75 micrometer I.D. fused silica capillary (PicoFrit, New Objective Inc., Woburn MA). Peptides were separated using a linear gradient from 2-85% buffer B
- SUBSTIT ⁇ TE SHEET (RULE 26) (Buffer A: 5% acetonitrile in water with 0.5% acetic acid and 0.005%, TFA; Buffer B: 80% acetonitrile, 10% n-propanol, 10%> water, with 0.5% acetic acid, 0.005% TFA).
- the LC effluent was electrosprayed directly into the sampling orifice of an LCQ DECA spectrometer (Thermo Finnigan, CA) using an adaption of the microscale electrospray interface[45].
- the LCQ DECA was operated to collect MS/MS spectra in a data dependent manner, with up to four of the most intense ions that exceeded a pre-set threshold being subjected to fragmentation and analysis.
- the MS/MS data generated was analyzed and matches to protein sequences in the NCBI non-redundant database (mammalian subset) were determined using both SEQUEST [46] and MASCOT [47] programs.
- Sequence identification was based on the Mowse score [48] (10> log(P), where P is the probability that the observed match found by the Mascot software is a random event). Protein scores greater than 60 were significant at p ⁇ 0.05. In each case, the predicted M r and pi of the identification matched the observed M r and pi values within
- SUBSTIT ⁇ TE SHEET (RULE 26) membrane topology of its putative Ca(2+)-dependent regulatory region. Biochim. Biophys. Acta, 1504(2-3):220-228 (2001).
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AU2002326644A AU2002326644A1 (en) | 2002-08-14 | 2002-08-14 | Method for isolating subpopulations of proteins that engage in protein-protein interactions |
US10/519,109 US20060275821A1 (en) | 2002-08-14 | 2002-08-14 | Method for isolating subpopulations of proteins that engage in protein-protein interactions |
PCT/US2002/025845 WO2004019967A1 (en) | 2002-08-14 | 2002-08-14 | Method for isolating subpopulations of proteins that engage in protein-protein interactions |
JP2004532531A JP2005535727A (en) | 2002-08-14 | 2002-08-14 | Method for isolating protein subpopulations involved in protein-protein interactions |
CA002492324A CA2492324A1 (en) | 2002-08-14 | 2002-08-14 | Method for isolating subpopulations of proteins that engage in protein-protein interactions |
EP02761370A EP1556065A4 (en) | 2002-08-14 | 2002-08-14 | Method for isolating subpopulations of proteins that engage in protein-protein interactions |
CN02829465.3A CN1658892A (en) | 2002-08-14 | 2002-08-14 | Method for isolating subpopulations of proteins that engage in protein-protein interactions |
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JP (1) | JP2005535727A (en) |
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WO2017207460A1 (en) * | 2016-05-30 | 2017-12-07 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | Ligand identification by co-fractionation |
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Citations (1)
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US5817789A (en) * | 1995-06-06 | 1998-10-06 | Transkaryotic Therapies, Inc. | Chimeric proteins for use in transport of a selected substance into cells |
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2002
- 2002-08-14 JP JP2004532531A patent/JP2005535727A/en not_active Withdrawn
- 2002-08-14 CN CN02829465.3A patent/CN1658892A/en active Pending
- 2002-08-14 EP EP02761370A patent/EP1556065A4/en not_active Withdrawn
- 2002-08-14 AU AU2002326644A patent/AU2002326644A1/en not_active Abandoned
- 2002-08-14 CA CA002492324A patent/CA2492324A1/en not_active Abandoned
- 2002-08-14 WO PCT/US2002/025845 patent/WO2004019967A1/en not_active Application Discontinuation
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US5817789A (en) * | 1995-06-06 | 1998-10-06 | Transkaryotic Therapies, Inc. | Chimeric proteins for use in transport of a selected substance into cells |
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Cited By (2)
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WO2017207460A1 (en) * | 2016-05-30 | 2017-12-07 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | Ligand identification by co-fractionation |
US10976310B2 (en) | 2016-05-30 | 2021-04-13 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | Ligand identification by co-fractionation |
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JP2005535727A (en) | 2005-11-24 |
CA2492324A1 (en) | 2004-03-11 |
CN1658892A (en) | 2005-08-24 |
EP1556065A1 (en) | 2005-07-27 |
EP1556065A4 (en) | 2005-10-19 |
AU2002326644A1 (en) | 2004-03-19 |
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