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WO2007083154A1 - Conjugués polymères amphipathiques pour la solubilisation de protéines hydrophobes - Google Patents

Conjugués polymères amphipathiques pour la solubilisation de protéines hydrophobes Download PDF

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WO2007083154A1
WO2007083154A1 PCT/GB2007/000208 GB2007000208W WO2007083154A1 WO 2007083154 A1 WO2007083154 A1 WO 2007083154A1 GB 2007000208 W GB2007000208 W GB 2007000208W WO 2007083154 A1 WO2007083154 A1 WO 2007083154A1
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composition
polymer
pluronic
protein
hydrophobic
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Siddhartha Ghose
William Bains
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Babraham Research Campus Ltd
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Babraham Bioscience Technologies Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • 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
    • C07K1/02—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding

Definitions

  • the invention relates to a hydrophobic protein solubilisation composition comprising an amphipathic polymer conjugated to a binding agent, to processes for preparing the composition and to the processes and uses of the composition in the solubilisation and isolation of hydrophobic proteins, particularly but not exclusively hydrophobic membrane proteins.
  • Tan, J. F. et al. (2005) Biomacromolecules 6, 498-506 describes the association behaviour of a series of biotinylated and non-biotinylated polymers (e.g. poly (ethylene oxide) -b-poly (2- (diethylamino) ethyl methacrylate) .
  • biotinylated and non-biotinylated polymers e.g. poly (ethylene oxide) -b-poly (2- (diethylamino) ethyl methacrylate
  • a protein solubilisation composition comprising an amphipathic polymer conjugated to a binding agent.
  • hydrophobic protein solubilisation composition comprising an amphipathic •polymer conjugated to a binding agent.
  • hydrophobic protein will be understood to refer to a protein whose stability and/or function is enhanced by its being at least partly embedded or immersed in an environment with a substantially lower dielectric constant than water, such as an apolar solvent or a lipid membrane.
  • a substantially lower dielectric constant than water such as an apolar solvent or a lipid membrane.
  • proteins are often found associated with, or partially or completely embedded in, the lipid membranes of cells and their organelles. Such proteins are often referred to as membrane proteins.
  • the hydrophobic protein is a hydrophobic membrane protein.
  • the protein is an integral or peripheral hydrophobic membrane protein.
  • amphipathic polymer refers to a polymer which has been prepared from hydrophilic and hydrophobic monomers. Thus, an amphipathic molecule will generally comprise both hydrophilic and hydrophobic components.
  • Non-limiting examples of hydrophilic mononieric components present in the amphipathic polymer include acrylic acid, acrylamide, methacrylic acid, styrene sulfonates, vinyl imidazole, vinyl pyrrolidone, poly (ethylene glycol) acrylates and methacrylates, dimethylaminoethyl methacrylate, diethyl amino ethyl methacrylate, t-butylaniinoethyl methacrylate, dimethylaminoethyl acrylate, diethylaminoethyl acrylate, methacrylamide, dimethyl acrylamide, dimethylaminopropyl methacrylamide, ethylene glycol methacrylate phosphate, ethylene oxide, 2-(methacryloyloxy)ethyl phthalate, 2-(methacryloyloxy)ethyl succinate, 3-sulfopropyl methacrylate and 3-sulfopropyl acrylate.
  • Protected monomers that generate acrylic or methacrylic acid after removal of the protecting group may also be used.
  • Suitable protected monomers include trimethylsilyl methacrylate, triniethylsilyl acrylate, 1-butoxyethyl methacrylate, 1-ethoxyethyl methacrylate, 1-butoxyethyl acrylate, 1- ethoxyethyl acrylate, 2-tetrahydropyranyl acrylate, 2-tetrahydropyranyl methacrylate, t-butyl methacrylate, t-butyl acrylate, methyl oxymethacrylate and vinyl benzoic acid.
  • the hydrophilic monomelic component present in the amphipathic polymer is ethylene oxide.
  • the hydrophilic components of the polymer will generally be present in amounts of 30%-99% by weight, suitably 40%-90% (e.g. 70%-85%) by weight of the composition.
  • Non-limiting examples of hydrophobic monomeric components present in amphipathic polymers include C 1-20 alkyl derivatives (e.g. alkyl oxides such as propylene oxide or hydroxyalkyls such as hydroxyalkyl acrylates and methacrylates (e.g. 2-(diethylamino)ethyl methacrylate (DEAEMA)) ,
  • C 2-20 alkenyl derivatives e.g. butadiene
  • cycloalkyl derivatives e.g. cycloalkyl acrylates and methacrylates
  • aromatic hydrocarbon and alkylsilyl groups
  • the hydrophobic monomeric component present in the amphipathic polymer is propylene oxide.
  • the hydrophobic components of the polymer will generally be present in amounts of l%-70% by weight, suitably 5%-55% (e.g. 15%-30%) by weight of the composition.
  • amphipathic polymer is a PluronicTM or SynperonicTM block co-polymer which are both alkene oxide co-polymers of ethylene oxide (e.g. polyethylene oxide; PEO) and propylene oxide (e.g. polypropylene oxide; PPO) as defined by the following formula:
  • Non-limiting examples of SynperonicTM block co-polymers include F108, L121 , L122, NPlO, NP30, P105, P85, P94, PE F68, PE L61 and PE L64 all of which are commonly available (Sigma- Aldrich, 3050 Spruce St. , St. Louis, MO 63103) . Additional examples of SynperonicTM block copolymers include L35, L-44, L-43, L-42, L-61 and L-31 (Boulares et al. , Pure and Applied Chemistry 70, 1239-1244) .
  • amphipathic polymer e.g. the PluronicTM and SynperonicTM block co-polymers
  • amphipathic polymer e.g. the PluronicTM and SynperonicTM block co-polymers
  • the amphipathic polymer e.g. the PluronicTM and SynperonicTM block co-polymers
  • these polymers therefore provide the advantage of solubilising the membrane proteins without denaturing them.
  • Solubilisation of membrane proteins is well known and has been performed using strong detergents (e.g. sodium dodecyl sulfate (SDS)) , however, such solubilisation is typically accompanied by complete denaturation of the membrane protein.
  • SDS sodium dodecyl sulfate
  • amphipathic polymer advantageously provides a stabilising effect by forming a polymer micelle incorporating the captured protein and is capable of retaining the protein's native conformation and function. Retention of the captured protein in a native condition provides the advantage of allowing efficient and accurate downstream characterisation of the captured protein (e.g. NMR analysis and binding studies) .
  • the amphipathic polymer additionally comprises one or more cross-linking agents.
  • Non-limiting examples of cross-linking agents include any monomer with polymerizable di- or polyfunctional groups, such as ethylene glycol dimethacrylate, pentaerythritol tetraacrylate, pentaerythritol triacrylate, 3-(acryloyloxy)-2-hydroxypropyl methacrylate, ethyleneglycol dimethacrylamide, mono-2-
  • Cross linking agents may be present within the amphipathic polymer in an amount of 0.1%-5% by weight of the total composition. In one embodiment, the amphipathic polymer contains 1% cross linking agent by weight.
  • references to “conjugated” include compositions wherein the binding agent is covalently attached to the amphipathic polymer. It is also envisaged that the binding agent may be incorporated into the polymer during polymerisation. It will also be appreciated that the binding agent may be conjugated to the polymer following polymerisation using a variety of techniques commonly known to the skilled person.
  • binding agent refers to an agent, component or moiety which is capable of specifically binding to a further agent, component or moiety or a group of structurally related agents, components or moieties.
  • the binding agent is one half of a binding pair.
  • binding pairs include streptavidin and avidin which both specifically bind to biotin.
  • Binding pairs may comprise "proteinaceous” and "non-proteinaceous” agents (e.g. both halves of the binding pair may be proteinaceous, both halves of the binding pair may be non-proteinaceous , or one half of the binding pair may be proteinaceous and the other half of the binding pair may be non-proteinaceous) .
  • streptavidin and avidin are proteinaceous binding agents which are one half of a binding pair which binds to the non-proteinaceous binding agent biotin.
  • proteinaceous binding agents include soluble globular proteins, such as wheat-germ agglutinin and glutathione-S-transf erase (GST) .
  • non-proteinaceous binding agents include glutathione and N-acetyl-D-glucosamine.
  • wheat-germ agglutinin is a proteinaceous binding agent which binds to the non-proteinaceous binding agent N-acetyl-D-glucosamine.
  • glutathione-S-transferase is a proteinaceous binding agent which binds to the non-proteinaceous binding agent glutathione.
  • one half of the binding pair may be a metal-chelating group (e.g. an organic metal-chelating group such as nitrilotriacetic acid) incorporated within the amphipathic polymer.
  • the metal-chelating group will then be charged with a metal ion, such as nickel) and the other half of the binding pair may be a protein with a metal- affinity tag (e.g. a histidine tag) .
  • a metal- affinity tag e.g. a histidine tag
  • the presence of the binding agent (e.g. one half of a binding pair) in the composition of the invention provides the key advantage of enabling the simple, one-step isolation of a specific protein from a solubilised extract containing a pool of proteins.
  • the composition allows a one- step isolation of a specific membrane protein or a group of membrane proteins from a solubilised extract containing a pool of hydrophobic membrane proteins.
  • the polymeric component of the composition is capable of solubilising a hydrophobic protein (e.g. a hydrophobic membrane protein) , however, this protein will be present with other proteins in the extract.
  • the isolation process subsequently involves the use of the other half of the binding pair to isolate the polymer-protein complex.
  • the other half of the binding pair will have affinity for the binding agent component of the composition of the invention to isolate the complex from the mixture. Therefore, the composition of the invention eliminates the need for complicated and time consuming purification procedures to isolate a given protein.
  • amphipathic polymer will depend upon the protein desired to be isolated (e.g. the hydrophobicity of the protein) .
  • the skilled person will take account of variables such as: number of transmembrane domains (if any) , percentage of hydrophobic amino acids in the protein sequence and percentage of surface amino acids in the protein sequence. Examples of how the hydrophobicity may be measured are described herein and include standard known procedures, such as Kyte-Doolittle or Hopps-Wood plots, inter alia . The skilled person would then prepare a hydrophobicity plot and select an amphipathic polymer accordingly.
  • references to "protein” include proteins, peptides, polypeptides and oligopeptides. Proteins may be synthetic or naturally occurring, and may be obtained by chemical synthesis, or by recombinant or non-recombinant methods. The protein may be produced using DNA recombination or mutation techniques. The protein may be produced in vivo in a whole animal, or in a eukaryotic or prokaryotic cell; alternatively, the protein may be generated using an in vitro method such as cell-free in vitro translation e.g. using E. coli lysate, wheat germ extract, or rabbit reticulocyte. Cell free in vitro translation methods can be employed following in vitro transcription, e.g. following phage or ribosome display.
  • in vitro method such as cell-free in vitro translation e.g. using E. coli lysate, wheat germ extract, or rabbit reticulocyte. Cell free in vitro translation methods can be employed following in vitro transcription, e.g. following phage or
  • a process for preparing a protein solubilisation composition as hereinbefore defined which comprises the step of:
  • Step (a) typically comprises reaction of an amphipathic polymer with a non-proteinaceous binding agent in the presence of suitable reagents (e.g. when the polymer is PluronicTM F127 or PluronicTM F68 and the binding agent is biotin, the reagents are typically l-(3-dimethylaminopropyl)-3 ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine, dissolved in N-methylpyrrolidinone) .
  • suitable reagents e.g. when the polymer is PluronicTM F127 or PluronicTM F68 and the binding agent is biotin, the reagents are typically l-(3-dimethylaminopropyl)-3 ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine, dissolved in N-methylpyrrolidinone
  • the processes described in schemes 1 and 2 may typically be performed in the presence of a suitable base (e.g. DMAP) and a suitable solvent (e.g. water soluble DIC) .
  • a suitable base e.g. DMAP
  • a suitable solvent e.g. water soluble DIC
  • an activation step (a) is required prior to conjugating the proteinaceous binding agent to the amphipathic polymer.
  • Suitable activating agents include succinic acid or derivatives thereof (e.g. N, N- disuccinimidyl carbonate; DSC) .
  • Step (a) typically comprises reacting the amphipathic polymer with a suitable activating agent (e.g. when the polymer is PluronicTM F127 the activating agent may be N,N-disuccinimidyl carbonate; DSC) in the presence of a suitable base (e.g. when the polymer is PluronicTM Fl 27 the base is 4-dimethylaminopyridine) and a suitable solvent (e.g. when the polymer is PluronicTM F127 the solvent is dioxane) .
  • a suitable activating agent e.g. when the polymer is PluronicTM F127 the activating agent may be N,N-disuccinimidyl carbonate; DSC
  • a suitable base e.g. when the polymer is PluronicTM Fl 27 the base is 4-dimethylaminopyridine
  • a suitable solvent e.g. when the polymer is PluronicTM F127 the solvent is dioxane
  • Step (b) typically comprises incubation of the activated polymer obtained in step (a) with the proteinaceous binding agent in the presence of a suitable buffer (e.g. when the polymer is PluronicTM F 127 and the binding agent is streptavidin, the buffer is PBS) at a suitable temperature (e.g. when the polymer is PluronicTM Fl 27 and the binding agent is streptavidin, the reaction is performed at room temperature) .
  • a suitable buffer e.g. when the polymer is PluronicTM F 127 and the binding agent is streptavidin, the buffer is PBS
  • a suitable temperature e.g. when the polymer is PluronicTM Fl 27 and the binding agent is streptavidin, the reaction is performed at room temperature
  • step (b) requires at least 10:1 ratio of activated amphipathic polymer to proteinaceous binding agent and that the proteinaceous binding agent has at least one exposed sulfhydryl (e.g. cysteine) on its folded surface.
  • sulfhydryl e.g. cysteine
  • activation of PluronicTM Fl 27 and F68 with succinic acid may be performed in accordance with schemes 3 and 4, respectively, below:
  • amphipathic block co-polymer is a block alkene oxide co-polymer. In a further embodiment, the amphipathic block copolymer is a PluronicTM or SynperonicTM block co-polymer.
  • a PluronicTM or SynperonicTM block co-polymer as hereinbefore defined, in the solubilisation of a hydrophobic protein.
  • the hydrophobic protein is a hydrophobic membrane protein.
  • the PluronicTM or SynperonicTM block co-polymer is PluronicTM F68 or PluronicTM F127.
  • Figure 1 demonstrates the results of SDS-PAGE analysis of a cell membrane solubilised by PluronicTM polymers, TritonTM-X and water as control;
  • Figure 2 demonstrates the results of SDS-PAGE analysis of biotinylated and unbiotinylated PluronicTM polymers in the presence of streptavidin;
  • FIG. 3 demonstrates the NMR spectra of biotinylated PluronicTM
  • Figure 4 demonstrates the results of SDS-PAGE analysis showing albumin recovery using biotinylated and unbiotinylated PluronicTM polymers followed by isolation using streptavidin;
  • Figures 5 and 6 demonstrate the results of SDS-PAGE analysis showing the effects of incubating activated PluronicTM F127 with wheat-germ agglutinin and glutathione-S-transf erase, respectively.
  • the cells were harvested by centrifugation at 5,000 rpm for 5 min (4 0 C) in a JLA 10,500 rotor in a Beckman Avanti J20-XP centrifuge, washed with water and pelleted by centrifugation as before.
  • the cells were resuspended in 100 ml ice-cold JR lysis buffer (20 mM Hepes.KOH pH 7.5, 50 mM KOAc pH 7.4, 0.1 M sorbitol, 2 mM EDTA, 1 mM DTT, 1 mM PMSF) and the cell walls disrupted by vortexing in the presence of glass beads (212-300 ⁇ m, Sigma, G9143) for 2 x 5 min.
  • Unbroken cells and cell debris were collected by centrifugation at 5,000 rpm for 10 min (4 0 C) in a JA 25.5 rotor in a Beckman Avanti J20-XP centrifuge and the supernate retained. Cells not collected by the previous step were collected by centrifugation at 7,000 rpm for 10 min (4 0 C) (in the previous rotor and centrifuge) .
  • the supernate was diluted in an equal volume of JR lysis buffer and microsomal membranes collected by centrifugation at 17,000 rpm for 30 min (4 0 C) (in the JA 25.5 rotor in the Beckman Avanti centrifuge) .
  • Membranes were resuspended in 10 ml buffer 88 (20 mM Hepes.KOH pH 7.5, 150 mM KOAc pH 7.4, 250 mM sorbitol) and re-pelleted by centrifugation at 17,000 rpm for 20 min (4 0 C) (in the JA 25.5 rotor in the Beckman Avanti centrifuge) .
  • Membrane pellet was resuspended in 0.45 ml membrane storage buffer (20 mM Hepes.KOH pH 7.4, 50 mM KOAc pH 7.4, 250 mM sorbitol, 1 mM DTT) , snap frozen in liquid nitrogen and stored at -8O 0 C.
  • Microsomal membranes were thawed on ice and aliquoted equally between 5 sample tubes (90 ⁇ l each) .
  • the membranes were solubilised using the following polymers/detergents at the concentrations shown, to a total volume of 200 ⁇ l:
  • the prepared solubilised samples were incubated on a tube roller (50 rpm) for 2 hr at room temperature to allow equilibration between membrane protein and polymer/detergent. After incubation, half of the material was centrifuged for 10 min at 13,000rpm at room temperature in a bench-top microfuge (Heraeus) to pellet insoluble membranous material and insoluble protein. The supernate was removed and precipitated by trichloroacetic acid (TCA) precipitation (add TCA to a final concentration of 15% (w/v) and incubated on ice for 15 min followed by centrifugation at 13,000 rpm for 10 min in a bench-top microfuge.
  • TCA trichloroacetic acid
  • TCA pellets were washed with 100 ⁇ l acetone and re-pelleted as before) .
  • the insoluble membranes and protein pellet was resuspended in 50 ⁇ l reducing sample buffer (2% (w/v) SDS, 80 mM Tris pH 6.8, 10% (v/v) glycerol, 1O mM EDTA pH 8.0, 0.001% (w/v) bromophenol blue, 100 mM DTT) and boiled (100 0 C for 5 min).
  • Precipitated material was resuspended in 20 ⁇ l reducing sample buffer and boiled (100 0 C for 5 min) . Both sets of samples (e.g.
  • Example 7 Preparation of Biotinylated-PluronicTM F68 (E7) PluronicTM F68 (Sigma, P5556) was supplied as a 10% (w/v) solution. PluronicTM F68 required freeze-drying because the biotinylation process will not tolerate water in the reaction mixture.
  • the biotinylation reaction was prepared in an analogous manner to Example 6 by dissolving the following components in approximately 4 ml NMP:
  • Example 8 Characterisation of Biotinylated-PluronicTM F127 (E6) and Biotinylated-PluronicTM F68 (E7) Multiple samples of 125 ⁇ g streptavidin were mixed with 150 ⁇ g Biotinylated-PluronicTM F127 (which may be prepared as described in E6) or PluronicTM F127, Biotinylated-PluronicTM F68 (which may be prepared as described in E7) or PluronicTM F68. These samples were incubated on ice for 20 min before 25 ⁇ l non-reducing sample buffer and 50 ⁇ l water were added. Samples were incubated at room temperature for 5 min and 5 ⁇ l of the following samples were loaded on a reducing SDS-PAGE gel which can be seen in Figure 2.
  • Biotinylated-PluronicTM F127 was also subjected to NMR analysis and the results of this analysis may be seen in Figure 3.
  • the peaks have been labelled with letters (e.g. a, b, c, d, e, f, g, h, i and j for Biotinylated-PluronicTM F127 (E6) in Figure 3 to associate a given peak with a given part of the biotinylated molecule.
  • Example 9 Isolation of Albumin using Biotinylated-PluronicTM Fl 27 (E6)
  • a Nunc maxi-sorp 96-well plate (Sigma, M9410) was coated with 500 ⁇ g/ml streptavidin (Invitrogen, S-888) , in PBS pH 8.0 overnight at 4 0 C. Streptavidin was removed and the wells blocked for non-specific interactions with 0.2% (w/v) MarvelTM milk powder in PBS pH 8.0 for 2 hr at room temperature with gentle agitation.
  • Conjugating a proteinaceous binding agent to a polymer first involves the step of activating the polymer. This procedure involved a one-step carbonate activation. The reaction was set up as follows:
  • PluronicTM F127, DSC and DMAP were weighed into the same vial and dissolved in 15 ml dioxane. They were mixed with stirring overnight at room temperature under nitrogen in a fume hood. The mixture was added slowly to ⁇ 200 ml diethyl ether while stirring to precipitate the polymer. The ether was decanted after the polymer precipitate was allowed to settle and rewashed with another 200 ml diethyl ether. The second wash was removed, the polymer dried by rotary evaporation and the last of the ether removed under vacuum. The polymer was immediately transferred to a weighed glass vial, sealed and stored at -2O 0 C. Approximately 4 g of polymer was recovered from the activation reaction.
  • Example 11 Conjugating Activated PluronicTM Fl 27 (ElO) to Wheat- Germ Agglutinin 10 mg of wheat-germ agglutinin (Sigma, L9640) was dissolved and equilibrated in 1 ml PBS pH 8.0. 100 mg activated PluronicTM Fl 27 (which may be prepared as described in ElO) was added to the mixture and was then dissolved. The conjugation reaction was left at room temperature to incubate for 4 hr. Isolation of conjugated ElO- wheat-germ agglutinin was undertaken using N-acetyl-D-glucosamine conjugated to agarose beads (Sigma, A2278) .
  • the arrow indicates the presence of free agglutinin in lane 4.
  • agglutinin e.g. a change in pi value due to conjugation of agglutinin to ElO
  • Example 12 Conjugating Activated PluronicTM F127 (ElO) to Glutathione-S-Transferase (GST)
  • This conjugation reaction was performed in an analogous manner to that described in Ell with the exception that 10 mg glutathione-S-transferase (GST; Sigma, G6511) was dissolved and equilibrated in 1 ml PBS pH 8.0 and isolation of conjugated ElO-GST was undertaken using glutathione conjugated sepharose beads (Amersham, 17-0756-01) .

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Abstract

La présente invention concerne une préparation de solubilisation de protéines hydrophobes comprenant un polymère amphipathique conjugué à un agent liant, ainsi que des procédés d'élaboration de la préparation et les procédés et utilisations de la préparation pour la solubilisation et l'isolement de protéines hydrophobes, en particulier, mais de façon non exclusive, des protéines membranaires hydrophobes.
PCT/GB2007/000208 2006-01-21 2007-01-22 Conjugués polymères amphipathiques pour la solubilisation de protéines hydrophobes Ceased WO2007083154A1 (fr)

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WO2009077046A1 (fr) * 2007-12-15 2009-06-25 Merck Patent Gmbh Procédé d'extraction de protéines membranaires

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WO2011004158A1 (fr) * 2009-07-08 2011-01-13 The University Of Birmingham Solubilisation de protéines membranaires

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