GB2434366A

GB2434366A - Composition for solubilisation of a hydrophobic protein

Info

Publication number: GB2434366A
Application number: GB0701153A
Authority: GB
Inventors: Siddhartha Ghose; William Bains
Original assignee: BABRAHAM BIOSCIENCE TECHNOLOGI
Current assignee: BABRAHAM BIOSCIENCE TECHNOLOGI
Priority date: 2006-01-21
Filing date: 2007-01-22
Publication date: 2007-07-25
Also published as: GB0601251D0; WO2007083154A1; GB0701153D0

Abstract

A composition for the solubilisation of a hydrophobic protein, particularly a membrane protein, is disclosed. The composition comprises an amphipathic polymer conjugated to a binding agent. The amphipathic molecule preferably comprises a hydrophilic monomeric component, such as ethylene oxide, and a hydrophobic monomeric component, such as propylene oxide. The binding agent is one half of a binding pair, such as streptavidin, avidin, biotin, wheat-germ agglutinin or glutathione-s-transferase. Processes for preparing the composition are also disclosed. A further embodiment provides the use of a polyoxypropylene-polyoxyethylene block co-polymer in the solubilisation of a hydrophobic protein.

Description

HYDROPHOBIC PROTEIN SOLUBILISATION COMPOSITION

FIELD OF THE INVENTION

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.

BACKGROUND OF THE INVENTION

Tan, J.F. et at. (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). * .

Lin, J. J. et a!. (2004) Langmuir 20, 5493-5500 describes an adhesiveness study of the effect of polymer chain length of polymersomes comprising a diblock copolymer of poly(ethylene oxide)-polybutadiene (PEO-PBD). S. S * S I * S

*: 20 US 6,087,452 (University of Utah) describes a modified surfactant compound having at least one polyethylene oxide (PEO) attached at a first end to at least one polypropylene oxide (PPO) block, with at least one of the PEO block having an organic metal-chelating end group attached to a second end.

US 5,696,090 (Xoma Corporation) describes a pharmaceutical composition comprising a polypeptide and a poloxamer surfactant which is claimed to have improved stability and resistance to aggregation, particle formation and precipitation.

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Tribet, C. et a!. (1996) Proc. Nati. Acad. Sci. USA 93, 15047-15050 describes the use of amphipol polymers in the solubilisation of membrane proteins in aqueous solutions.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided a protein solubilisation composition comprising an amphipathic polymer conjugated to a binding agent.

According to a further aspect of the invention, there is provided a hydrophobic protein solubilisation composition comprising an amphipathic polymer conjugated to a binding agent.

DETAILED DESCRIPTION OF THE INVENTION * **s

It will be appreciated that references to "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. In vivo, such 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.

In one embodiment, the hydrophobic protein is a hydrophobic membrane protein. In a further embodiment, the protein is an integral or peripheral hydrophobic membrane protein.

There are a variety of ways of measuring the degree of hydrophobicity of a protein, which include (but are not limited to) the retention time of the protein on hydrophobic chromatographic media, the ability to partition the protein onto or into a model lipid membrane (such as a Langmuir-Blodgett

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film), the aggregate, average or other integral of the individual hydrophobicities of the component amino acids of the protein (as defined by their partition coefficient between water and octanol -called LogP), or the presence of regions of such amino acids defining extended hydrophobic regions in the proteins, as determined by methods such as the Kyte-Doolittle or Hopps-Wood plots.

It will be appreciated that references to "amphipathic polymer" refer 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 monomeric 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, diethylaminoethyl methacrylate, t-butylaminoethyl methacrylate, dimethylaminoethyl acrylate, diethylaminoethyl acrylate, :. * methacrylamide, dimethylacryl amide, 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, trimethylsilyl acryl ate, 1 -butoxyethyl methacryl ate, 1 -ethoxyethyl methacrylate, 1 -butoxyethyl acryl ate, 1-ethoxyethyl acrylate, 2-tetrahydropyranyl acrylate, 2-tetrahydropyranyl methacrylate, t-butyl methacrylate, t-butyl acryl ate, methyl oxymethacrylate and vinyl benzoic acid.

In one embodiment, the hydrophilic monomeric 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 C120 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)), C220 alkenyl derivatives (e.g. butadiene), cycloalkyl derivatives (e.g. cycloalkyl acrylates and methacrylates), aromatic hydrocarbon and :*. alkylsilyl groups. * . ***.

In one embodiment, 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 1%-70% by weight, suitably 5%-55% (e.g. 15%-30%) by weight of the composition.

In one embodiment, the 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: HO(CZH4O)a(CH6O)b(C2H4O)aH Non-limiting examples of PluronicTM block co-polymers include L44NF (a=12, b=20), F68 (a=78, b=30), F68NF (a=80, b=27), P85 (a=26, b39), F87NF (a64, b=37), F1O8NF (a=141, b=44), F127 (a=100, b=65) and F127 (a=101, b=56), 6100, 3100, 4300 and all are commonly available (BASF Aktiengesellschaft, Carl-Bosch-Str. 38, 67056 Ludwigshafen, Germany). Additional examples of PluronicTM block co-polymers include P123, F88 and F98. In a further embodiment, the amphipathic polymer is PluronicTM F68 or PluronicTM F127.

Non-limiting examples of SynperonicTM block co-polymers include F108, L121, L122, NP1O, NP3O, P105, P85, P94, FE F68, PE L61 and FE L64 all of which are commonly available (Sigma-Aldrich, 3050 Spruce St., St. Louis, MO 63103). Additional examples of SynperonicTM block co-polymers include L35, L-44, L-43, L-42, L-61 and L-31 (Boulares et al., Pure and Applied Chemistry 70, 1239-1244). * *..

The presence of the amphipathic polymer (e.g. the PluroniCTM and SynperonicTM block co-polymers) in the composition of the invention provides the key advantage of solubilising hydrophobic proteins and in * I particular, solubilising hydrophobic membrane proteins from within the environment of the cell membrane complex. 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 denaturaion of the membrane protein. By contrast, the 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).

In one embodiment, 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 methacryl ate, ethyleneglycol dimethacrylamide, mono-2- (methacryloyloxyethyl) maleate, divinyl benzene, or other monomers with polymerizable di-or polyfunctional groups.

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.

It will be appreciated that 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.

It will be appreciated that references to "binding agent" refer 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. In one embodiment, the binding agent is one half of a binding pair. Examples of such 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). Thus, streptavidin and avidin are proteinaceous binding agents which are one half of a binding pair which binds to the non-proteinaceous binding agent biotin.

Further examples of proteinaceous binding agents include soluble globular proteins, such as wheat-germ aggluti nm and gI utathione-S-transferase (GST). Further examples of non-proteinaceous binding agents include glutathione and N-acetyl-D-glucosamine. Thus, in one embodiment, wheat-germ agglutinin is a proteinaceous binding agent which binds to the non-proteinaceous binding agent N-acetyl-D-glucosamine. In an alternative embodiment, glutathione-S-transferase is a proteinaceous :. binding agent which binds to the non-proteinaceous binding agent :.. ::: 15 glutathione. S...

In one embodiment, 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 *5S*SS * the binding pair may be a protein with a metal-affinity tag (e.g. a histidine tag). The use of such binding agents and their incorporation into polymers are described in US 6,087 452, the contents of which are herein incorporated by reference.

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. In particular, the composition allows a one-step isolation of a specific membrane protein or a group of membrane proteins from a sot ubilised extract containing a pooi of hydrophobic

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membrane proteins. For example, as mentioned above, 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. For example, 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.

It will be appreciated that the choice of amphipathic polymer will depend :. upon the protein desired to be isolated (e.g. the hydrophobicity of the protein). For example, 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 * 20 standard known procedures, such as Kyte-Doolittle or Hopps-Wood plots, I.....

* inter alia. The skilled person would then prepare a hydrophobicity plot and select an amphipathic polymer accordingly.

It will be appreciated that 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.

According to a further aspect of the invention, there is provided a use of a protein solubilisation composition as hereinbefore defined in the isolation of a protein.

According to a further aspect of the invention, there is provided a use of a protein solubilisation composition as hereinbefore defined in the isolation of a hydrophobic protein.

According to a further aspect of the invention, there is provided a process for preparing a protein solubilisation composition as hereinbefore defined which comprises the step of: *... . . . . (a) conjugating an amphipathic polymer as hereinbefore defined with a non-proteinaceous binding agent as hereinbefore defined. S..

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*0 Step (a) typically comprises reaction of an amphipathic polymer with a * 20 non-proteinaceous binding agent in the presence of suitable reagents (e.g. *5*S** * when the polymer is PluronicTM F127 or PluronicTM F68 and the binding agent is biotin, the reagents are typically 1-(3-dimethylaminopropyl)-3 ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine, dissolved in N-methylpyrrolidinone).

For example, conjugation of PluronicTM F127 and F68 with biotin may be performed in accordance with schemes 1 and 2, respectively, below: _ HjJ5IH PE" PD00Er ---______ ocr oor ocr, --- 1UV b 1(A) * -1 u.i........---- -S F127 Scheme 1 H N/k N H PEO78PP0PEO76 PEOl8PP03oPEO7__..1c/ F68 o Scheme 2 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. * ii water soluble DIC). * *

According to a further aspect of the invention, there is provided a process for preparing a protein solubilisation composition as hereinbefore defined which comprises the steps of: (a) activating an amphipathic polymer as hereinbefore defined; * * (b) conjugating the activated amphipathic polymer with a proteinaceous binding agent as hereinbefore defined.

Thus, prior to conjugating the proteinaceous binding agent to the amphipathic polymer, an activation step (a) is required. 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 F127 the base is 4-dimethylaminopyridine) and 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 F127 and the binding agent is streptavidin, the buffer is PBS) at a suitable temperature (e.g. when the polymer is PluronicTM F127 and the binding agent is streptavidin, the reaction is performed at room temperature).

Generally, 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 suithydryl (e.g. cysteine) on its folded surface. S. * * ** * *S

For example, activation of PluronicTM F127 and F68 with succinic acid may be performed in accordance with schemes 3 and 4, respectively, below: *...S. * *

PEO,PPOa,PEO, Ff27 0 Scheme 3 PE0,6PP0PE078 - PEO?3PPO3OPEOThJL, Scheme 4 As a further example, activation of PluronicTM F127 and F68 with DSC may be performed in accordance with schemes 5 and 6, respectively, below: PE0100PP065PE0100 PEO100PPO65PEO10O F127 0 Scheme 5 PE078PP030PE078 PE078PP030PEO780 Scheme 6 * * According to a further aspect of the invention, there is provided a use of *..* an amphipathic block copolymer as hereinbefore defined in the solubilisation of a protein. * U..

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In one embodiment, the amphipathic block co-polymer is a block alkene : 15 oxide co-polymer. In a further embodiment, the amphipathic block co- * 5 polymer is a PluronicTM or SynperonicTM block co-polymer.

According to a further aspect of the invention, there is provided a use of a PluronicTM or SynperonicTM block co-polymer as hereinbefore defined, in the solubilisation of a hydrophobic protein. In one embodiment, the hydrophobic protein is a hydrophobic membrane protein. In one embodiment, the PluronicTM or SynperonicTM block co-polymer is PluronicTM F68 or PluronicTM F127.

The invention will now be illustrated with reference to the following non-limiting Figures and Examples in which: Figure 1 demonstrates the results of SDS-PAGE analysis of a cell membrane solubilised by PIuronicTM polymers, TritonTMX and water as control; Figure 2 demonstrates the results of SDS-PAGE analysis of biotinylated and unbiotinylated PluronicTM polymers in the presence of streptavidin; Figure 3 demonstrates the NMR spectra of biotinylated PluronicTM F127; S. * S :... 15 Figure 4 demonstrates the results of SDS-PAGE analysis showing albumin recovery using biotinylated and unbiotinylated PluronicTM polymers followed by isolation using streptavidin; and 5'.

Figures 5 and 6 demonstrate the results of SDS-PAGE analysis showing the effects of incubating activated PluronicTM F127 with S.....

* wheat-germ agglutinin and glutathione-S-transferase, respectively.

Examples 1-5: Extraction of Microsomal Membrane Proteins Using Amphipathic Polymers Preparation of Yeast Microsomal Membranes 11(500 ml per 2 1 flask) of Saccharomyces cerevisiae diploid strain W303 (National Collection of Yeast Cultures) culture was grown to saturation (0D600 2-3) overnight in pre-autoclaved YPD (1% (w/v) yeast extract (Melford,Y1333), 2% (wlv) peptone (Melford, P1328), 2% (w/v) glucose (Sigma, 47829) at 30 C with shaking at 200 rpm. The cells were harvested by centrifugation at 5,000 rpm for 5 mm (4 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 jim, Sigma, G9143) for 2 x 5 mm.

Unbroken cells and cell debris were collected by centrifugation at 5,000 rpm for 10 mm (4 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 mm (4 C) (in the previous rotor and centrifuge).

The supernate was diluted in an equal volume of JR lysis buffer and microsonial membranes collected by centrifugation at 17,000 rpm for 30 mm (4 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 mm (4 C) (in the JA 25.5 rotor in the *.Is..

* 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 -80 C.

Extraction from Microsomal Membranes Microsomal membranes were thawed on ice and aliquoted equally between sample tubes (90 jil each). The membranes were solubilised using the following polymers/detergents at the concentrations shown, to a total volume of 200 jil: Example Polymer/Detergent Supplier (Product Number Code) El 2% (w/v) PluronicTM Fl27 Sigma (P2443) E2 2% (w/v) PluronicTM F68 Sigma (P5556) E3 2% (w/v) SynperonicTM Sigma (86214-5 g) P94 E4 MilliQ water N/A E5 1% (w/v) TritonTM-X-iOO Sigma (T9284) 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 :. 5 centrifuged for 10 mm 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 trichioroacetic acid (TCA) precipitation (add TCA to a final concentration of 15% (wlv) and incubated on ice for 15 mm followed by centrifugation at 13,000 rpm for 10 mm in a bench-top microfuge. TCA pellets were : washed with 100.tl acetone and re-pelleted as before). The insoluble * membranes and protein pellet was resuspended in 50 tl reducing sample buffer (2% (w/v) SDS, 80 mM Tris pH 6.8, 10% (vlv) glycerol, 10 mM EDTA pH 8.0, 0.001% (w/v) bromophenol blue, 100 mM DTT) and boiled (100 C for 5 mm). Precipitated material was resuspended in 20 j.tl reducing sample buffer and boiled (100 C for 5 mm). Both sets of samples (e.g. supernatant and membrane pellet fractions) were then subjected to analysis by SDS-PAGE. 10 tl of each of the samples were run on a 4- 10% (w/v) NuPage Bis-Tris 12-well gel (Invitrogen, NPO3O2BOX) until the dye front had reached the bottom of the gel. The gel was coomassie stained for 20 mm and destained overnight with gentle agitation. The results of the SDS-PAGE may be seen in Figure 1 wherein the lane marked with an "M" refers to the markers (5 il PageRulerTM markers; Helena Biosciences; SM0671). It is apparent from Figure 1 that one particular protein (between the 130 and 100 kDa markers) was more efficiently solubilised by the polymer of El than those of the polymers of E2, E3 and the control (E4). Furthermore, the polymer of E3 solubilised a protein (larger than the 170 kDa marker) more effectively than the polymers of El and E2. It can also be seen in the membrane pellet fractions that the polymers of E2 and E3 were more effective at solubilising one particular protein (between the 170 kDa and 130 kDa markers) by virtue of the absence of this protein when compared with the polymer of El. Thus, Figure 1 demonstrates key differences in the pattern of proteins extracted from the membranes between the polymers of El, E2 and E3. * . * S..

:::* 15 Example 6: Preparation of Biotinylated-PluronicTM F127 (E6) PluronicTM F127 (Sigma, P2443) was supplied solid and was used directly * in the following Biotinylation reaction:

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PluroniclM F127 FW Mass No. of moles Equivalents Biotinylation (E6) (gil) Biotin (Sigma, B4501) 244.3 100 mg 4.1x104 1 PluronicTM F127 12,600 5.2 g 4.1x104 1 DMAP (Sigma, 122.17 50 mg 4.1x104 1 D5640) Wat. Sol. DIC 191.71 157 mg 8.2x104 2 (Sigma, E7750) The above components were dissolved in approximately 5.5 ml NMP (Sigma, 328634) followed by incubation overnight with gentle stirring at room temperature in a fume hood.

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Removal of solvent was carried out by filtration using a Vivascience Vivaflow filtration pump using a membrane with a 5,000 Da MWCO.

Filtration was carried Out at a pump setting of 2.5-3 for approximately 48 hours at 4 C with the solvent being exchanged for distilled water.

Completion of solvent exchange was followed by freeze-drying of the polymer to obtain solid biotinylated material (E6).

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 S...

*,** 15 Example 6 by dissolving the following components in approximately 4 ml NMP: * . S * *5

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PluronictM F68 FW (gil) Mass No. of Equivalents Biotinylation (E7) moles *:" Biotin 244.3 100 mg 4.1x104 1 PluronicTM F68 8,400 3.44 g 4.1x104 1 DMAP 122.17 50 mg 4.1x104 1 Wat. So!. DIC 191.71 157 mg 8.2x104 2 Solvent exchange and freeze-drying was performed as in Example 6 to obtain solid biotinylated material (E7).

Example 8: Characterisation of Biotinylated-Pluronic F127 (E6) and Biotinylated-PluronicTM F68 (E7)

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Multiple samples of 125 j.ig streptavidin were mixed with 150 tg Biotinylated-PluronicTM F127 (which may be prepared as described in E6) or Ph'ronicTM F127. BiotinvlatedPluronicTM F68 (which may be prepared as described in E7) or PluronicTM F68. These samples were incubated on ice for 20 mm before 25 j.il non-reducing sample buffer and 50.tl water were added. Samples were incubated at room temperature for 5 mm and 5 il of the following samples were loaded on a reducing SDS-PAGE gel which can be seen in Figure 2.

Lane Contents M 5pi Broad range markers (Bio-Rad; 161-0138) 1 125.tg streptavidin + 150.tg Biotinylated-PluronicTM F127 in 100.tl :.. ::: 2 125.Lg streptavidin + 150 g Biotinylated-PluronicTM F68 in 100 jil *:*. 3 125 j.tg streptavidin + 150.tg PluronicTM F127 in 100 eq. * .tl

:. * 4 125.tg streptavidin + 150.tg PluronicTM F68 in 100 i.tl * C 125 ig streptavidin It can be seen from Figure 2 that a difference exists between biotinylated polymer and un-biotinylated polymer interacting with streptavidin.

Streptavidin in the presence of un-biotinytaced polymer (lanes 3 and 4) appears exactly the same as streptavidin alone (lane 5), whereas lanes 1 and 2 show a marked difference. This difference is likely to be indicative that biotinylation of the polymers has occurred and that this material is capable of binding to streptavidin.

Biotinylated-PluronicTM F127 (E6) was also subjected to NMR analysis and the results of this analysis may be seen in Figure 3. In the NMR spectra, 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 toassociate a given peak with a given part of the biotinylated molecule.

These characterisation experiments clearly demonstrate the successful conjugation of biotin to PluronicTM F127 (E6) and PluronicTM F68 (E7).

Example 9: Isolation of Albumin using Biotinylated-PluronicTM F127 (E6) A Nunc maxi-sorp 96-well plate (Sigma, M9410) was coated with 500 j.tg/ml streptavidin (Invitrogen, S-888), in PBS pH 8.0 overnight at 4 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. *... * S

*.. 15 Albumin (1.5 mg) was associated with 3% (w/v) PluronicTM F127, Biotinylated PluronicT4 F127 (which may be prepared as described in E6), PluronicTM F68 and Biotinylated PluronicTM F68 (which may be prepared * as described in E7) by incubation at room temperature for 1 hr with * 20 frequent inversion. Samples (maximum volume 250 l.Ll) were incubated in coated and blocked wells for 2 hr at room temperature with gentle agitation. Samples were removed (unbound material) and the wells washed once with 1% (w/v) PluronicTM F127, Biotinylated PluronicTM F127, PluronicTM F68 or Biotinylated PluronicTM F68, respectively. Bound protein-polymer complexes were eluted by addition of 200 l reducing sample buffer to the wells and heating at 95 C.

SDS-PAGE analysis was then conducted using the following samples and the results may be seen in Figure 4: Lane Contents M 5tl Broad range markers (Bio-Rad; 161-0138) U Unbound material (7.5p.1) 1 PluronicTM F127 associated with albumin (15tl) 2 Biotinylated-PluronicTM F127 associated with albumin (15tl) 3 PluronicTM F68 associated with albumin (15tl) 4 Biotinylated-PluronicTM F68 associated with albumin (1 5j.tl) It can be seen from Figure 4 that more protein was recovered in the presence of biotinylated polymer (lanes 2 and 4) than without (lanes 1 and :. 3). The presence of albumin after removal of unbound material is due to aggregation on the plate which could be removed with more stringent washing conditions. This analysis demonstrated that proteins such as albumin can be isolated using a pair of binding partners when one of those partners (e.g. biotin) is conjugated to a polymer of the invention. * * S

* * 10 Example 10: Activation of PlurOnieTM F127 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: Material MW Mass/mg Vol/ No. of moles Equiv. ml

PluronicTM F127 12600 5000 N/A 0.000396825397 1 DSC (Sigma, 256.17 101.66 N/A 0.000396825397 1 43720) DMAP 122.17 24.24 N/A 0.000198412698 0.5 Dioxane (Sigma, N/A N/A 15 N/A N/A 296309) PlljrnnicTM 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 -20 C. Approximately 4 g of polymer was recovered from the activation reaction.

:... Example 11: Conjugating Activated PluronicTM F127 (ElO) to Wheat-S...

Germ Agglutinin :.; 15 10 mg of wheat-germ agglutinin (Sigma, L9640) was dissolved and equilibrated in 1 ml PBS pH 8.0. 100 mg activated PluronicTM F127 :. (which may be prepared as described inElO) was added to the mixture * and was then dissolved. The con3ugatlon 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).

p1 of reducing sample buffer was added to 50 j.tl of the conjugated sample and subjected to SDS-PAGE analysis using the follow samples: Lane Contents M 5j.tl PageRulerTM Markers (Helena Biosciences; SM0671) 1 Conjugated ElO-wheat-germ agglutinin (15p1) 2 Blank 3 Blank 4 Agglutinin only (50tg) The gel was run for 90 mm at 150V, Coomassie stained for 20 mm and de-stained overnight with gentle agitation and results may be observed in Figure 5.

The arrow indicates the presence of free agglutinin in lane 4. When compared with the corresponding position in lane 1 (shown by the "} symbol) it is apparent that incubating wheat-germ agglutinin with ElO in solution has changed the mobility on the SDS-PAGE gel. This difference is likely to be due to a change in agglutinin (e.g. a change in p1 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 Eli 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 E1O-GST was undertaken using glutathione conjugated sepharose beads (Amersham, 17-0756-01).

SDS-PAGE analysis was performed in an identical manner to that described in Eli using the follow samples: Lane Contents M 5p.1 Markl2TM Markers (Invitrogen; LC5677) 1 Conjugated Ei0-GST (10iil) 2 1% (w/v) ElO only (lOpi) 3 GST only (5tg) 4 GST only (10tg) 5t1 PageRulerTM Markers (Helena Biosciences; SM0671) The results shown in Figure 6 demonstrate a difference between GST incubated with ElO (lane 1) and GST alone (lanes 3 and 4). As with Example 11, this is likely to be due to a change in GST (e.g. a change in p1 value due to conjugation of GST to ElO).

Abbreviations DIC 1-(3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride DMAP 4-dimethylaminopyridine DSC N,N-disuccinimidyl carbonate NMP N-methylpyrrolidinone

S

S..... PEO polyethylene oxide *:*. PPO polypropylene oxide * 15

S S. * * * S * S

*..*** S * * I

Claims

CLAIMS

1. A hydrophobic protein solubilisation composition comprising an amphipathic polymer conjugated to a binding agent.

2. A composition as defined in claim 1, wherein the protein is a hydrophobic membrane protein.

3. A composition as defined in claim 1 or claim 2, wherein the amphipathic molecule comprises both hydrophilic and hydrophobic monomeric components.

4. A composition as defined in claim 3, wherein the hydrophilic monomeric component is ethylene oxide.

5. A composition as defined in claim 3 or claim 4, wherein the hydrophilic components are present in amounts of 30%-99% by weight.

6. A composition as defined in any of claims 3 to 5, wherein the hydrophobic monomeric components include C1.20 alkyl derivatives (e.g. alkyl oxides such as propylene oxide or hydroxyalkyls such as hydroxyalkyl acrylates and methacrylates), cycloalkyl derivatives (e.g. cycloalkyl acrylates and methacrylates), aromatic hydrocarbon and alkylsilyl groups.

7. A composition as defined in claim 6, wherein the hydrophobic monomeric component is propylene oxide.

8. A composition as defined in any of claims 3 to 7, wherein the hydrophobic components are present in amounts 1%-70% by weight.

9. A composition as defined in any preceding claims, wherein the amphipathic polymer is a PIuronic or Synperonic block co-polymer.

10. A composition as defined in claim 9, wherein the PluronicTh block co-polymer polymer is Pluronic F68 or Pluronic F127.

11. A composition as defined in any preceding claims, wherein the binding agent is one half of a binding pair.

12. A composition as defined in claim 11, wherein the binding agent is selected from streptavidin, avidin, biotin, soluble globular proteins (such as wheat-germ agglutinin or glutathione-S-transferase (OST)), glutathione, N-acetyl glucosamine, a metal chelating group (such as nitrilotriacetic :... acid charged with a metal ion, such as nickel) and a metal-affinity tag 15 (such as a histidine tag). * .*

13. Use of a composition as defined in any preceding claims in the isolation of a hydrophobic protein. S. S * . . * *

: 20 14. A process for preparing a protein solubilisation composition as defined in any of claims 1 to 12, which comprises the step of: (a) conjugating an amphipathic polymer as defined in any of claims 3 to 10 with a non-proteinaceous binding agent.

15. A process for preparing a protein solubilisation composition as defined in any of claims 1 to 12, which comprises the steps of: (a) activating an amphipathic polymer as defined in any of claims 3 to 10; and (b) conjugating the activated amphipathic polymer with a proteinaceous binding agent.

16. Use of a P1uronic or Synperonic block co-polymer, in the solubilisation of a hydrophobic protein.

17. Use as defined in claim 16, wherein the protein is a hydrophobic membrane protein.

18. Use as defined in claim 16 or claim 17, wherein the Pluronic block co-polymer polymer is Pluronicl'M F68 or P1uronic F127. S. * . * *e. * * **** *5 5 * * S * S. S..

S *5 S * S S * . SSS'

S S