Much effort is currently concentrated on research devoted to biofunctional surface modification. In particular, patterned surfaces, where different areas expose different functions, are fundamental to the development of microarrays for highthroughput gene and protein analyses. DNA microarrays have proved very successfulell, and the concept is in the process of being applied to protein arrays However, in contrast to DNA fragments, proteins are easily denatured in contact with a solid support, and the straight-forward robotic printing of proteins onto chemically reactive glass slides will not necessarily be applicable as a generic protocol for the preparation of protein arrays.

A first requirement for non-denaturing conditions for immobilized proteins, and thus efficient protein microarray matrices, is high resistance of the underlying support towards non-specific protein adsorption. This requirement can be met by using supported lipid bilayers (SPBs), for example, phosphatidylcholine lipid bilayers, as supports for protein immobilisation using, e. g., biotin-streptavidint7'g, NTA-Ni2+-His9j, or maleimide-thiol coupting [lol. Patterning of SPBs may be achieved I'], and even combined with protein immobilisation, utilising microfluidics devices, or using dispensing and spontaneous fusion of vesicles with reconstituted membrane proteins into spots of supported lipid bilayers at the solid: air interface t12l. Thus, lipid membrane and/or vesicle arrays may be formed locally using dispensed vesicle solutions, arrays of lipid membranes with varying lipid composition achieved through micro contact printing, or gradients in microfluidic flow devices. However, these techniques have their problems, typically associated with the construction and operation of microfluidic devices as well as solvent evaporation during dispensing.

Niemeyer et al. E131 describe the use of complementary DNA (cDNA) strands to direct spatial distribution of DNA-labelled proteins to a DNA array.

However, the technique of Niemeyer et aL 1131 has the disadvantage of bringing the DNA-labelled proteins into close contact with the solid support, thereby increasing the likelihood of denaturation and non-specific protein adsorption.

There is therefore a need for a technique that provides the advantages of low protein denaturation/high specific protein adsorption without loosing the advantages of a the improved spatial distribution technique described by Niemeyer et aL Accordingly, a first aspect of the present invention provides for the use of a lipid vesicle having bound thereto a targeting polynucleotide and a protein, polypeptide or peptide, which targeting polynucleotide is complementary to a region of a scaffold polynucleotide, to spatially distribute the protein, polypeptide or peptide at a defined position relative to the scaffold polynucleotide.

This use is appropriate as an alternative to planar SPBs where electrical access to both sides of the membrane is not required. It provides for improved spatial distribution of the protein, polypeptide or peptide in comparison to prior art techniques using planar SPBs, since spatial control can be achieved without the need for dispensing or microfluidic devices.

The present invention also provides, as a second aspect, a method for immobilizing a protein, polypeptide or peptide comprising the steps of (a) providing, as a first component, an immobilised scaffold polynucleotide; (b) providing, as a second component, a lipid vesicle having bound thereto a targeting polynucleotide and a protein, polypeptide or peptide, which targeting polynucleotide is complementary to a region of a scaffold polynucleotide ; and (c) contacting the first and second components such that the targeting polynucleotide binds to the scaffold polynucleotide ; thereby to produce an immobilised protein, polypeptide or peptide.

The step of contacting the first and second components such that the targeting polynucleotide binds to the scaffold polynucleotide is performed under conditions that substantially avoids non-specific binding of targeting polynucleotide to non-matching scaffold polynucleotide.

Thus an immobilised protein, polypeptide or peptide is produced, i. e. immobilised via the lipid vesicle, through binding of the targeting polynucleotide binds to the scaffold polynucleotide.

Accordingly, the present invention also provides a system for spatial distribution of proteins, polypeptides or peptides comprising a scaffold polynucleotide and a lipid vesicle having bound thereto a targeting polynucleotide and a protein, polypeptide or peptide, which targeting polynucleotide is complementary to a region of the single stranded scaffold polynucleotide.

A method according to the second aspect of the present invention may optionally further comprising the steps (d) of providing, as a third component, an agent of interest; (e) contacting the third component with the protein, polypeptide or peptide either before or after step (c); and (f) detecting whether the agent of interest has interacted with the protein, polypeptide or peptide; thereby to determine whether the immobilised protein, polypeptide or peptide is able to specifically interact with the agent of interest.

The agent of interest may be any suitable molecule. It may, for example, be a naturally occurring or synthetic protein, polypeptide or peptide; it may be a polynucleotide or oligonucleotide; it may be a carbohydrate; it may be a lipid; it may be a pharmaceutical compound; it may be a signalling molecule; it may be a cell surface protein, such a cell surface protein that is characteristic of a particular cell or tissue type, or the surface protein of a pathogen, a tumour cell, a virally infected cell or the like.

The immobilised protein, polypeptide or peptide is assayed for its ability to interact with the agent of interest. For example, the immobilised protein, polypeptide or peptide may bind covalently or non-covalently with the agent of interest. Alternatively, the immobilised protein, polypeptide or peptide may react with the agent of interest to cause a chemical modification to the agent of interest and/or to the immobilised protein, polypeptide or peptide.

Detection of such interaction may be performed by methods known in the art and will typically involve array scanning.

It may be advantageous contacting the third component with the protein, polypeptide or peptide before step (c), e. g. by mixing the functionalised vesicles with the agent of interest in solution, in order to allow for an equilibrium of interaction to be reached in a shorter time than would be required if the agent is only allowed to interact with the protein, polypeptide or peptide after step (c). Moreover, contacting the third component with the protein, polypeptide or peptide before step (c) can help further reduce any interaction of the protein, polypeptide or peptide or agent with the solid phase on which the scaffold polynucleotide is immobilised.

The third component may comprise, a plurality of different agents of interest, such as up to 10,20, 30,40, 50,60, 70,80, 90,100 or more different agents of interest.

The method of the second aspect of the present invention may further comprising the steps of (g) identifying the protein, polypeptide or peptide that specifically interacts with the agent of interest; and (h) producing further protein, polypeptide or peptide having a sequence derived from the protein, polypeptide or peptide that specifically interacts with the agent of interest, which further protein, polypeptide or peptide retains the ability to specifically interact with the agent of interest.

The step of identifying the protein, polypeptide or peptide that specifically interacts with the agent of interest may involve sequencing the protein, polypeptide or peptide or may involve identifying the clone that it was generated from. In the second option, the clone can be further analysed to determine the nature of the protein, polypeptide or peptide, e. g. by analysis of the protein, polypeptide or peptide produced by the clone, or by analysis of the encoding polynucleotide held by the clone.

Following the identifying step, protein, polypeptide or peptide having a sequence derived from the protein, polypeptide or peptide that specifically interacts with the agent of interest may be produced.

By"having a sequence derived from"is included the meaning of fragments and variants of the identified protein, polypeptide or peptide. A fragment is less than 100% of the whole protein, polypeptide or peptide, e. g. 90, 80, 70, 60,50, 40,30% or less. A variant may be prepared by techniques well known in the art, such as to enhance or reduce the immunogenicity of the protein, polypeptide or peptide. For example, a few amino acid residues may be changed. Thus a variant may have one or more positions where there have been amino acid insertions, deletions, or substitutions, either conservative or non-conservative, provided that such changes result in a protein, polypeptide or peptide for which the ability to specifically interact with the agent of <BR> <BR> interest has not significantly been changed. "Significantly"in this context means that one skilled in the art would say that the properties of the variant may still be different but would not be unobvious over the ones of the original protein, polypeptide or peptide.

By"conservative substitutions"is intended combinations such as Gly, Ala; Val, Ile, Leu; Asp, Glu ; Asn, Gln ; Ser, Thr; Lys, Arg; and Phe, Tyr. Such variants may be made using the methods of protein engineering and site- directed mutagenesis known in the art, such as those disclosed in US Patent No 4,302, 386 issued 24 November 1981 to Stevens, incorporated herein by reference.

Typically a variant will have more than 40%, usually at least 50%, more typically at least 60%, preferably at least 70%, more preferably at least 80%, yet more preferably at least 90%, even more preferably at least 95%, most preferably at least 98% or more sequence identity with protein, polypeptide or peptide from which it was derived. The percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequence has been aligned optimally. The alignment may alternatively be carried out using the Clustal W program (Thompson et al., 1994). The parameters used may be as follows: Fast pairwise alignment parameters: K-tuple (word) size; 1, window size; 5, gap penalty; 3, number of top diagonals; 5. Scoring method: x percent. Multiple alignment parameters: gap open penalty; 10, gap extension penalty; 0.05. Scoring matrix: BLOSUM.

Therefore, a third aspect of the present invention also provides a protein, polypeptide or peptide obtainable by the method of the second aspect of the present invention, which protein, polypeptide or peptide has the ability to specifically interact with an agent of interest.

Optionally, a method according to the second aspect of the present invention may additionally comprise the step of formulating the further produced protein, polypeptide or peptide with a pharmaceutically acceptable carrier or diluent.

Methods for the production of lipid vesicles are well known in the art. For example, see Singh et al (2000, Anal. Chem., 72,6019-6024) and Alfonta et al (2001, Anal. Chem., 73,5287-5295).. The lipid vesicle is typically an intact vesicle. It may have the potential to act as a carrier of both water- soluble and transmembrane protein.

The targeting polynucleotide may be DNA, RNA, LNA or PNA, or a chimera of two of more of these, such as a chimera of DNA and LNA; a chimera of DNA and PNA; a chimera of PNA and LNA ; a chimera of RNA and LNA; a chimera of RNA and PNA; a chimera of RNA and DNA; a chimera of DNA, PNA and LNA; a chimera of DNA, RNA and LNA; a chimera of DNA, RNA and PNA; a chimera of RNA, PNA and LNA; or a chimera of DNA, RNA, PNA and LNA. Typically the targeting polynucleotide is DNA.

The targeting polynucleotide is typically single stranded. It may be any appropriate length. It may, for example, be between 10 and 200 bases in length, such as up to 150,100, 80,60, 50,40, 30 or 25 bases. A length of about 20 bases will usually be suitable.

The scaffold polynucleotide may be may be DNA, RNA, LNA or PNA, or a chimera of two of more of these, as defined above for the targeting polynucleotide. Typically the scaffold polynucleotide is DNA.

The scaffold polynucleotide is typically single stranded. It may be any appropriate length. It may, for example, be between 10 and 2000 bases in length or more, such as up to 1500,1000, 800,600, 500,400, 300,200, 150, 100,80, 60,50, 40,30 or 25 bases. A length of about 20 base pairs may be suitable.

The targeting polynucleotide is complementary to a region of a scaffold polynucleotide. By complementary is meant that the scaffold polynucleotide is able to bind to the targeting polynucleotide under stringent <BR> <BR> conditions. "Stringent conditions", as defined herein, refers to conditions that operate to allow the targeting polynucleotide to bind substantially exclusively to the scaffold polynucleotide. For example, the conditions may be of low ionic strength and high temperature for washing, for example 0. 1X SSC, 0.2% SDS (65-70°C, although more moderately stringent conditions, such as 0.2X SSC, 0.1% SDS @ 58-65°C may be included in this definition. Typically the term complementary means that the at least a region of the targeting polynucleotide has a complementary sequence to a region of the scaffold polynucleotide. The regions of complementarity are typically at least 10,15, 20,25, 30 or more bases in length. It may be useful to use a targeting polynucleotide with absolute homology to the scaffold polynucleotide.

A scaffold polynucleotide may contain one or more portion of complementarity, i. e. regions that are complementary to a targeting polynucleotide ; it may also contain one or more regions complementary to different targeting polynucleotides: this can allow a number of identical or non-identical targeting polynucleotides to anneal to the scaffold polynucleotide in a defined and patterned manner. In such cases, the effect may be to bring multiple proteins (bound to the targeting polynucleotide via the lipid vesicle) into a defined spatial relationship with one another.

Thus, in one embodiment, the scaffold polynucleotide comprises multiple portions of complementarity. The multiple portions of complementarity may be the same or different.

The scaffold polynucleotide may be immobilized on any solid phase and in any pattern with multiple identical or different scaffold polynucleotides.

For example, the scaffold polynucleotide may be part of an array, such as an array containing multiple'spots' (i. e. discrete areas), each spot having a different scaffold polynucleotide, or homogeneous population thereof, immobilised to it.

It may be beneficial to the utility of the invention for the array to have a defined pattern and distribution of scaffold polynucleotide, such that a correlation can be made between the position of a given scaffold polynucleotide on the array and its sequence.

Thus in one embodiment, the scaffold polynucleotide is part of a polynucleotide array, which, depending on the nature of the scaffold polynucleotide, may be a DNA array. Typically, the array will be a cDNA microarray.

As discussed above, DNA microarrays are known in the art E'l. New technology, called VLSIPSTM, has enabled the production of extremely small chips that contain hundreds of thousands or more of different molecular probes. These biological chips or arrays have probes arranged in arrays, each probe assigned a specific location. Biological chips have been produced in which each location has a scale of, for example, ten microns.

Scanning devices can be used to examine each location in the array and determine whether an interaction of interest has occurred at any given location on the chip.

The targeting polynucleotide may be bound to lipid vesicle by any method known in the art. For example, it may be derivatised at the 3'or 5'end with a lipid-binding, or lipophilic molecule; it may be derivatised at the 3'or 5' end with a moiety that has a binding affinity for a molecule that has been attached to the lipid vesicle. Thus the targeting polynucleotide may be derivatised with biotin, cholesterol or fluorescein.

The vesicle may have more than one targeting polynucleotide. It may have, for example, 2,3, 4, 5 or more targeting polynucleotides. Preferably, the vesicle is homogeneous for targeting polynucleotides.

The protein, polypeptide or peptide may comprise the sequence of any natural or artificial sequence. It may, for example, comprise the sequence of a truncated naturally occurring protein, or be a fusion protein (i. e. comprise sequences derived from two naturally occurring proteins that are not normally joined together). Typically the protein, polypeptide or peptide is a water-soluble protein or a transmembrane protein. A transmembrane protein will typically comprise a hydrophobic domain that will anchor in a lipid-bilayer.

A common water-soluble protein suitable for use in the present invention is an antibody. An antibody, as defined herein, may have the sequence of any suitable naturally occurring or synthetically produced antibody. Antibodies having a sequence based on naturally occurring non-human antibodies may be "humanised". For example, the variable domains of non-human origin antibodies may be fused to constant domains of human origin (Morrison et al (1984) Proc. Natl. Acad Sci. USA 81, 6851-6855).

The term"antibody"as used herein includes antibody fragments. An antibody fragment will typically include at least one of the variable heavy (VH) and variable light (VL) domains, which are involved in antigen recognition. Common antibody fragments include Fab-like molecules (Better et al (1988) Science 240,1041) ; Fv molecules (Skerra et al (1988) Science 240,1038) ; single-chain Fv (scFv) molecules where the VH and VL partner domains are linked via a flexible oligopeptide (Bird et al (1988) Science 242, 423; Huston et al (1988) Proc. Natl. Acad. Sci. USA 85,5879) ; and single domain antibodies (dAbs) comprising isolated V domains (Ward et al (1989) Nature 341,544). A general review of the techniques involved in the synthesis of antibody fragments that retain their specific binding sites is to be found in Winter & Milstein (1991) Nature 349,293-299. Fab, Fv, ScFv, dAb and other antibody fragments can be manufactured recombinantly using techniques known in the art, such as by expression in and secretion from E. coli. Other antibody fragments include F (ab') 2 fragments, which are "bivalent", i. e. have two antigen combining sites. In contrast, Fab, Fv, ScFv and dAb fragments are monovalent, having only one antigen combining sites.

The protein, polypeptide or peptide may be derived from a library such as an antibody library as defined in WO 01/83734, Clackson et al., 1991, Nature 352 : 624-628; Marks et al., 1991, J Mol. Biol. 222: 581-597), peptide libraries (see Smith, 1985, Science 228: 1315-1317), expressed cDNA libraries (see Santi et al., 2000, J. Mol. Biol. 296: 497-508), libraries on other scaffolds than the antibody framework such as affibodies (see Gunneriusson et al., 1999, Appl. Environ. Microbiol. 65: 4134-40) the contents of which are incorporated herein by reference.

The molecular libraries may be expressed in vivo in prokaryotic (see Clackson et al., 1991, Nature 352: 624-628; Marks et al., 1991, R Mol. Biol. 222: 581- 597) or eukaryotic (see Kieke et al., 1999, Proc. Natl. Acad. Sci. USA 96: 5651-6) cells or may be expressed in vitro without involvement of cells (see Hanes & Pluckthun, 1997, Proc. Natl. Acad. Sci. USA 94: 4937-42; He & Taussig, 1997, Nucleic Acids Res. 25: 5132-4; Nemoto et al., 1997, FEBS Lett 414: 405-8).

In cases where the protein, polypeptide or peptide is obtained from a protein based library, the genes encoding the library members potential binders (anti- ligands) are often packaged in viruses and the protein, polypeptide or peptide is displayed at the surface of the virus (see Clackson et al., 1991, Nature 352: 624-628; Marks et al., 1991, J. Mol. Biol. 222: 581-597 ; Smith, 1985, Science 228: 1315-1317). The most commonly used such system, today, is filamentous bacteriophage displaying antibody fragments at their surfaces, the antibody fragments being expressed as a fusion to the minor coat protein of the bacteriophage. However, also other systems for display using other viruses (EP 39578), bacteria (see Gunneriusson et al., 1999, Appl. Environ.

Microbiol. 65: 4134-40; Daugherty et al., 1998, Protein Eng. 11: 825-32; Daugherty et aL, 1999, Protein Eng. 12: 613-21), and yeast (see Shusta et al., 1999, J. Mol. Biol. 292: 949-56) have been used. In addition, recently, display systems utilising linkage of the polypeptide product to its encoding mRNA in so called ribosome display systems (see Hanes & Pluckthun, 1997, Proc. Natl.

Acad. Sci. USA 94: 4937-42; He & Taussig, 1997, Nucleic Acids Res. 25: 5132-4 ; Nemoto et al., 1997, FEBS Lett 414: 405-8), or alternatively linkage of the polypeptide product to the encoding DNA (see US Patent No. 5,856, 090 and WO 98/37186) have been presented.

Thus, a population of lipid vesicles, as defined above, may be provided, the population comprising different proteins, polypeptides or peptides bound to the lipid vesicles. The different proteins, polypeptides or peptides may be derived from a protein library, for example as defined above.

Typically the library has been'pre-selected'in order to reduce the library size. For example, a library as discussed above, can be assayed for a desirable property (e. g. if the library is an antibody library, it may be assayed for the ability to bind an antigen) using techniques known in the art, and putative successful candidates selected in order to reduce the number of candidate library members. The selected candidates can then be cloned separately. Each clone can be used to produce a protein, polypeptide or peptide candidate, which may then be bound to a lipid vesicle. Each type of lipid vesicle (i. e. typed by the identity of the protein, polypeptide or peptide candidate) can be bound to a unique targeting polynucleotide. Thus the identity of the targeting polynucleotide can be correlated with the identity of the clone candidate protein, polypeptide or peptide.

Thus, in one embodiment, each lipid vesicle is bound to only one type of protein, polypeptide or peptide. Each vesicle may have a unique protein, polypeptide or peptide bound thereto (i. e. unique within the vesicle population).

It may be beneficial for each type of vesicle to have a different targeting polynucleotide, such that each different protein, polypeptide or peptide is linked to its own unique targeting polynucleotide (via the lipid vesicle).

Therefore each type of lipid vesicle may be bound to a targeting polynucleotide unique within the lipid vesicle population.

The skilled user can produce a population of such vesicles, with the knowledge of which targeting polynucleotide is linked to which protein, polypeptide or peptide.

Therefore, there can be provided a lipid vesicle population as described above wherein there is a link between the phenotype of each vesicle (i. e. the property of its bound protein, polypeptide or peptide) and its unique targeting polynucleotide.

This can be useful in the uses and methods of the invention.

A fourth aspect of the present invention provides a method for detecting the presence of a target molecule comprising (a) providing, as a first component, a lipid vesicle having bound thereto a polynucleotide tag and a protein, polypeptide or peptide; (b) providing, as a second component, a sample suspected of containing the target molecule; (c) contacting the first and second components under conditions to allow the a protein, polypeptide or peptide to bind to any target molecule present in the sample; (d) removing unbound vesicle, without allowing for detrimental levels of non- specific binding; (e) amplifying the polynucleotide tag to produce an amplification product; and (f) detecting the amplification product; wherein the presence of the amplification product is indicative of the presence of the target molecule in the sample.

In the above case, a polynucleotide'tag'can be the same as a targeting polynucleotide as defined herein, although the sequence of the tag is not constrained by the need to bind to a scaffold polynucleotide.

The skilled person will appreciate that the step of removing unbound vesicle can be achieved by any suitable method known in the art, such as by using one or more washing steps.

The sample suspected of containing the target molecule may be any type of sample, although it is typically a biological sample, which may or may not have been processed. For example, it may be an ex vivo sample, such as a blood sample or tissue sample.

The step of amplifying the polynucleotide tag to produce an amplification product can be performed by methods well known in the art, such as by using the polymerase chain reaction (PCR) by providing appropriate oligonucleotides primers. The step of detecting the amplification product can be performed using routine methods and may, for example, involve size fractionation using gel chromatography and/or sequencing and/or hybridisation to a polynucleotide chip as described herein, to identify the amplification product.

Accordingly, a fifth aspect of the present invention provides for the use of a lipid vesicle having bound thereto a polynucleotide tag and a protein, polypeptide or peptide, as an amplifiable ligand for a target molecule.

In a method or use according to the third or fourth aspects of the present invention, the detection of a unique polynucleotide tag, can allow the skilled person to rapidly determine which type of lipid vesicle, and hence which protein, polypeptide or peptide, has bound to a sample. Therefore, the skilled person can identify which ligands are present in the sample assayed.

Moreover, in a use or method according to the first or second aspects of the present invention, the array of immobilised scaffold polynucleotide, as described above, can complement the utility of such a population of vesicles.

For example, where the skilled user is aware of the correlation between the position and the identity of each scaffold polynucleotide on the array, and between the phenotype and targeting polynucleotide of each vesicle in a population then, following the binding of the vesicle population to the array, positional information (e. g. position of detected binding or position of a detected reaction) can be used to determine the identity of a protein, polypeptide or peptide of interest.

Thus the protein, polypeptide or peptide, such as an antibody, can be spatially distributed at a defined position relative to the scaffold polynucleotide, and/or at defined positions on an array of such scaffold polynucleotides.

The identity may correspond to a known sequence. Alternatively, the identity may correspond with a known clone in a library of clones, thus allowing the user to refer back to the library, select the clone of interest and further characterise the protein, polypeptide or peptide it encodes.

In one embodiment of the present invention, polynucleotide library members, such as defined above, are compartmentalised in lipid vesicles using lipid- water emulsions. Typically each lipid vesicle contains only one polynucleotide library member, which is then translated in vitro, and the resultant protein, polypeptide or peptide incorporated in the lipid membrane, thereby displaying the phenotype on the surface of the vesicle (Tawfik and Griffiths, 1998, Nature Biotech., 16,652-656). This allows the phenotype to be linked to, and selected for, via the genotype, e. g. by assaying for the binding of vesicles to an antigen/ligand. Thus a lipid vesicle for use in the present invention may comprise a protein, polypeptide or peptide displayed on the vesicle surface using the method of Tawfik and Griffiths (op. cit.).

Accordingly, the present invention also provide a population of vesicles as defined above, each lipid vesicle containing a polynucleotide library member that encodes the protein, polypeptide or peptide bound to the surface of the vesicle.

Thus, a library as defined above may be screened for a desirable trait (e. g. if an antibody library is used, screen for binding to an antigen). This initial round of screening can be completed using methods known in the art and is intended to reduce the library size. Polynucleotide sequences encoding selected candidates can then be cloned, amplified and incorporated into lipid vesicles, followed by in vitro translation of the incorporated polynucleotide sequences to produce a population of lipid vesicles, each expressing a single candidate library member protein, polypeptide or peptide on its surface. The vesicles are each provided with a unique targeting polynucleotide as defined above, and immobilised on an array of complementary scaffold polynucleotides as defined above.

The resulting array of immobilised candidate library member proteins, polypeptides and/or peptides can be further screened to identify candidates with the most desired property, based on the array format of screening.

We have demonstrated the use of a protein-array as defined above by antigen capturing using His-tagged single chain antibody fragments (scFv) ["I linked to Ni2+-NTA-and DNA-modified lipid vesicles.

Furthermore, we have also demonstrated a DNA-directed array of different fluorescently labelled vesicles.

The present invention has the advantage over Niemeyer et at. of providing non-denaturing conditions for immobilized proteins, and thus efficient protein microarray matrices, through the high resistance of the underlying support towards non-specific protein adsorption.

The protein, polypeptide or peptide may be bound to lipid vesicle by any method known in the art. For example, a water-soluble protein, polypeptide or peptide such as, an antibody, may be His-tagged and the vesicle may be NTA-Ni2+ modified, as exemplified in the present application.

Alternatively, transmembrane proteins will naturally anchor in a lipid vesicle. Other options known the persons skilled in the art include chemical linkage of the protein, polypeptide or peptide to a lipid moiety or to a membrane-bound protein or fragment thereof, such as a membrane-spanning helix, or recombinant fusion of the protein, polypeptide or peptide to the sequence of a membrane-bound protein or fragment thereof, such as a membrane-spanning helix.

As discussed above, the further produced protein, polypeptide or peptide provided by a method according to the second aspect of the present invention may be used in the preparation of a pharmaceutical formulation in admixture with a pharmaceutically or veterinarily acceptable adjuvant, diluent or carrier.

Preferably, the formulation is a unit dosage containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof, of the active isolated further produced protein, polypeptide or peptide.

For the purposes of preparation of the formulation, the further produced protein, polypeptide or peptide may be provided in the form of a non-toxic organic, or inorganic, acid, or base, addition salt.

The formulation may be suitable for administration orally or by any parenteral route, in a pharmaceutically acceptable dosage form. Depending upon the disorder and patient to be treated, as well as the route of administration, the further produced protein, polypeptide or peptide may be administered at varying doses.

In human therapy, the further produced protein, polypeptide or peptide can be administered alone but will generally be administered in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.

For example, the further produced protein, polypeptide or peptide can be administered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed-or controlled- release applications. The further produced protein, polypeptide or peptide may also be administered via intracavernosal injection.

Such tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxy-propylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the isolated polypeptide may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.

The further produced protein, polypeptide or peptide can also be administered parenterally, for example, intravenously, intra-arterially, intraperitoneally, intrathecally, intraventricularly, intrasternally, intracranially, intra-muscularly or subcutaneously, or may be administered by infusion techniques. They are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary.

The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.

Formulations suitable for parenteral administration include aqueous and non- aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

For oral and parenteral administration to human patients, the daily dosage level of the isolated polypeptide will usually be from 1 to 1000 mg per adult (i. e. from about 0.015 to 15 mg/kg), administered in single or divided doses.

Thus, for example, the tablets or capsules of the compound of the invention may contain from 1 mg to 1000 mg of active compound for administration singly or two or more at a time, as appropriate. The physician in any event will determine the actual dosage which will be most suitable for any individual patient and it will vary with the age, weight and response of the particular patient. The above dosages are exemplary of the average case.

There can, of course, be individual instances where higher or lower dosage ranges are merited and such are within the scope of this invention.

The further produced protein, polypeptide or peptide can also be administered intranasally or by inhalation and is conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, e. g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoro-ethane, a hydrofluoroalkane such as 1,1, 1,2- tetrafluoroethane (HFA 134A3 or 1,1, 1,2, 3,3, 3-heptafluoropropane (HFA 227EA3), carbon dioxide or other suitable gas. In the case of a pressurised aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active compound, e. g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e. g. sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of a compound of the invention and a suitable powder base such as lactose or starch.

Aerosol or dry powder formulations are preferably arranged so that each metered dose or"puff'contains at least 1 mg of a further produced protein, polypeptide or peptide for delivery to the patient. It will be appreciated that he overall daily dose with an aerosol will vary from patient to patient, and may be administered in a single dose or, more usually, in divided doses throughout the day.

Alternatively, the further produced protein, polypeptide or peptide can be administered in the form of a suppository or pessary, or they may be applied topically in the form of a lotion, solution, cream, ointment or dusting powder. The further produced protein, polypeptide or peptide may also be transdermally administered, for example, by the use of a skin patch. It may also be administered by the ocular route, particularly for treating diseases of the eye.

For ophthalmic use, the further produced protein, polypeptide or peptide can be formulated as micronised suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride. Alternatively, it may be formulated in an ointment such as petrolatum.

For application topically to the skin, the further produced protein, polypeptide or peptide can be formulated as a suitable ointment containing the further produced protein, polypeptide or peptide suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, it can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

Formulations suitable for topical administration in the mouth include lozenges comprising the isolated polypeptide in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.

Generally, in humans, oral or topical administration of the further produced protein, polypeptide or peptide is the preferred route, being the most convenient. In circumstances where the recipient suffers from a swallowing disorder or from impairment of drug absorption after oral administration, the further produced protein, polypeptide or peptide may be administered parenterally, e. g. sublingually or buccally.

For veterinary use, a further produced protein, polypeptide or peptide is administered as a suitably acceptable formulation in accordance with normal veterinary practice and the veterinary surgeon will determine the dosing regimen and route of administration which will be most appropriate for a particular animal.

The invention will now be described in more detail by reference to the following non-limiting Figures and Examples.

Figure 1 shows a schematic representation of the substrate-directed surface modification protocol developed in the present study. Si02 and Au induce different surface modification, the discrimination resulting from differences in adsorption of (i) biotin-BSA and (ii) spontaneous fusion of lipid vesicles into a supported lipid bilayer on Si02, followed by (iii) neutravidin and (iv) biotin-DNA binding. In the more advanced protocol, steps (i) to (iv) were followed by (vi) scFv binding to (v) Ni2+-NTA-modified vesicles followed by (vii) specific binding of antigen CT (ß-subunit).

Figure 2 shows QCM-D data (Af, =3 (related to mass uptake) and ADn=3 <BR> <BR> (related to viscoelastic properties) ) from experiments using differently coated crystals: (a) Si02, and (b) Au. (i) biotin-BSA adsorption (10 g/ml) rendering low adsorption on SiOz (Af,=3 <2 Hz) compared with Au (ouf"=3 approx. 45 Hz). (ii) Formation of a SPB on Si02 (POPC vesicles, 20 g/mL) was verified from the characteristic initial Af and AD response, as observed previously'. The signature of vesicle rupture was not observed for the weak vesicles adsorption on the biotin-BSA coated Au.

(iii-iv) In the following steps, the two crystals were exposed, subsequently, to neutravidin (10 ug/mL) and biotin-cDNA (0.25 uM). Essentially no response was observed on the lipid bilayer-covered Si02, whereas significant binding of both neutravidin and biotin cDNAB occurred to the modified Au. (v) Eventually, POPC vesicles (15 pg/mL), doped with 0.5 % cholesterol-DNAB, bound preferentially on the cDNA-modified Au (see text).

Figure 3 shows microscope image of a SiO2-coated QCM crystal with spots of Au. The substrate was exposed to the same sequence of injections as in Figure 2, with the exception that (i) (cholesterol-cDNAB (25 nM) and (ii) fluorescein-DNAB (0.25 uM) were added prior to the addition NBD-dyed DNA-modified POPC vesicles. a) Excitation around 550 nm caused the rhodamine-dyed vesicles immobilised to the gold spots to fluoresce (>590 nm), as opposed to when b) shorter wavelengths (450-480 nm) were applied, causing instead the fluorescein-derivatised DNA fragments immobilized to the supported lipid bilayer on the Si02 to fluoresce (510-560 nm). c) Verification of highly specific binding of Cy3-labeled CT-ß (~100 amol per spot, estimated from QCM-D data) to scFv on Ni2+-NTA functionalized vesicles formed on the gold spots. Low non-specific was proven using QCM-D. d) The same substrate and filters as in a) -c), in which steps (i) to (iv) (Fig. 2) was followed by biotin-cDNAB injection (20 uM), through a thin capillary, leading to preferential binding to a single Au spot (to the right). NBD-dyed POPC vesicles doped with 0.5% of cholesterol-DNAB bound selectively to the cDNAB-modified spot (15 , ug/mL, >15'). An adjacent spot (to the left) was thereafter functionalized by biotin-cDNAA, added in excess (0.25 p. M, 5'), after which rhodamine-dyed POPC vesicles doped with 0.5% of cholesterol-DNAA (15 pg/mL, >15') were bound selectively to the cDNAA-modified spot. Scale bars: 100 urn.

Examples Materials and Methods Water was deionised and filtered (MilliQ unit, Millipore).

DNA strands (5'-TAG-TTG-TGA-CGT-ACA-CCC-CC-3' (DNAA); 5'- TAT-TTC-TGA-TGT-CCA-CCC-CC-3' (DNAB) ; 5'-TGT-ACG-TCA- CAA-CTA-CCC-CC-3' (cDNAA); 5'-TGG-ACA-TCA-GAA-ATA-CCC- CC-3' (cDNAB)), derivatised at the 3'-end with biotin (biotin-DNAA; biotin-DNAB), cholesterol (cholesterol-cDNAA ; cholesterol-DNAB ; cholesterol-cDNAB), or fluorescein (fluorescein-cDNAB) (MedProbe, Norway).

Stock solutions of DNA conjugates (20 uM in 10 mM Tris, 1 mM EDTA, pH 8.0) and proteins (biotin-amidocaproyl labelled BSA (Sigma, 1 mg/mL in water), neutravidin (Pierce, 1 mg/mL in buffer: 10 mM Tris, pH 8.0, 100 mM NaCI) were aliquote and stored at minus 20°C until use.

His-tagged scFv antibody (clone CT-17, 1 mg/ml in PBS, pH 7.4), kindly provided by BioInvent Therapeutics (Lund, Sweden) and-subunit cholera toxin (Sigma Chemical Co, St Louis, USA) labelled with Cy3 (Amersham Pharmacia Biotech, Uppsala, Sweden) were stored at +4°C until use.

1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (POPC, Avanti Polar Lipids, AL, USA) was dissolved in chloroform.

For fluorescent vesicles, 0.5 % (w/w) of Lissamine rhodamine B 1,2- dihexadecanoyl-sn-glycero-3-phosphoethanolamine (rhodamine-DHPE) (Molecular Probes, USA) or 2- (12- (7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino) dodecanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD- HPC) (Molecular Probes, USA) was added to the lipid solution.

For NTA-Ni2+ modified vesicles, 5% (w/w) of 1, 2-Dioleoyl-sn-Glycero-3- {[N (5-Amino-l-Caboxypentyl) iminodiAcetic Acid] succenyl} Dogs-NTA (Avanti Polar Lipids, AL, USA) was added to the lipid solution.

A lipid film was formed on the wall of a flask by evaporation of the solvent under N2 (> I h), and then hydrated with buffer (5 mg/mL).

Vesicles were prepared by extrusion (11 x through 0.1 Hm and 11X through 0. 03 um polycarbonate membranes (Whatman, USA) ), and stored at 4°C under N2.

DNA-labelling was achieved by addition of 0.5 % (w/w) of cholesterol- DNA to the vesicle solution (corresponding to approximately 3 DNA strands per vesicle), with 250mM imidazole present for the Ni2+-NTA- lipids.

All substrates (AT-cut quartz crystals, fO = 5 MHz, with either gold or Si02) and the QCM-D instrument (Q-sense D 300) were from Q-sense AB, Sweden.

The crystals were cleaned in 10 mM SDS (>15'), followed by 2xrinsing with water, drying (N2), and Lav-ozone treatment (10'). Si02-coated crystals were patterned by evaporation of 3 nm of Ti and 100 nm of Au through a mask.

Results In the present work, simple biofunctional patterns were prepared based on preferential protein adsorption and supported lipid bilayer formation on an Au/Si02 template. The critical initial discriminating step for the construction of the cDNA array was achieved under conditions where biotin-BSA adsorption on Au is significantly pronounced compared with biotin-BSA adsorption on SiO2 (preferential adsorption of proteins to one of two regions of a patterned substrate has been demonstrated previously (hydrophobic patches on Zu alternating platinum and Si02 regions22), but not as here combined with lipid bilayers). This was then followed by lipid bilayer formation on the bare Si02 substrates surrounding the biotin- BSA functionalized gold spots, subsequently followed by neutravidin binding to biotin-BSA (on Au), and binding of biotin-DNA to neutravidin (on Au) (steps i to iv in Fig. 1).

The protocol for the substrate-directed surface modification was established by QCM-D measurements using quartz crystals coated by either Si02 or Au (Fig. 2). Quartz crystal microbalance with dissipation monitoring (QCM-D) experiments are particularly useful to distinguish between adsorption and fusion of vesicles because of the simultaneous measurement of changes in resonance frequency, f (related to coupled mass), and energy dissipation, D (related to viscous losses in adsorbed films) [21, 211. All Of and AD values presented here were measured at the third overtone (15 MHz).

To achieve, eventually, cDNA-directed capturing of intact vesicles, cholesterol-DNA conjugates were added to the lipid vesicles (functionalized with Ni2+-NTA or not) resulting in the spontaneous anchoring of the cholesterol moiety into the hydrophobic region of the membrane (step v in Fig. 1 & 2).

While addition of cholesterol-DNA-modified vesicles to the SPB-coated Si02 (step v, Fig. 2a) resulted in modest changes in Afin=3 and ADn=3 (minus 20 Hz and 2X10-6, respectively), the mass-uptake (decrease in A/) was at least 15 times larger on the cDNA-modified Au (step v, Fig. 2b, see also figure legend).

This experiment was repeated on a patterned substrate (Au spots evaporated on SiO2), and the binding of dyed DNA-modified vesicles was imaged by fluorescence microscopy. Significantly, the Au spots fluoresced due to selective immobilisation of dyed vesicles, whereas very low background (non-specific) fluorescence was observed (Figure 3a). Figure 3b shows how another cholesterol derivatised DNA strand was anchored to the planar bilayer on Si02, and hybridised with a complementary, fluorescein-labelled strand, prior to the addition of the DNA-labelled vesicles, thus coexisting with the DNA-labelled vesicles on the Au substrate. The extension to patterns of functionalized DNA-labelled vesicles was proven by addition of His-tagged scFv (c. f. step vi, Fig. 1) and its fluorescently labelled antigen (cholera toxin sub unit B, CT-p) (c. f. step vi, Fig. 1) to DNA immobilized Ni2+-NTA-modified vesicles (Fig. 3c), demonstrating highly specific and sensitive detection of CT-P. Note in particular the low background, a prerequisite for extension towards large scale protein identification.

Finally, the applicability of the described surface modification for the preparation of arrays of different immobilized vesicles was proved (Fig.

3d), demonstrating the potential of the protocol towards surface directed sorting of differently functionalized vesicles.

In order to use cDNA arrays as a template for rapid sorting of a mixture of different DNA-labelled vesicles, exchange of DNA-cholesterol between different vesicles must be sufficiently low. The present protocol was not optimised in this respect. However, at a concentration of 20 nM, the coupling of cholesterol-labelled DNA to planar lipid bilayers and subsequent hybridisation was apparently irreversible and stable over hours (not shown).

Discussion In conclusion, we have presented a simple and novel surface preparation protocol for spontaneous formation of patterned supported lipid membranes, directed by the properties of the underlying solid inorganic support (Au/Si02), thus eliminating the need of stamping or microfluidics, as in previous strategies developed for the formation of hybrid surfaces of proteins and SPBs.

The choice of Au as the support for the functional spots was motivated from future surface plasmon resonance sensor applications, but as long as the biomacromolecules used in the initial discrimination step display relatively weak adsorption on Si02, it will be compatible with a large number of materials.

By introducing a cholesterol-based strategy for DNA-labelling of vesicles or supported lipid bilayers, it was further demonstrated that multifunctional patterns of lipid assemblies can be formed on top of protein/supported lipid bilayer patterns, here illustrated by (i) a pattern composed of immobilized DNA-labelled vesicles (on Au), proven compatible highly specific antigen detection utilising scFv antibodies, surrounded by an inert supported lipid bilayer (on SiO2), and (ii) surface-directed sorting of vesicles to two different cDNA-modified spots.

In light of future protein chips applications, the present protocol is compatible not only with water soluble proteins, as proven here, but also with transmembrane proteins, where the lipid environment and an aqueous surrounding are known to be extremely critical throughout the whole preparation protocol. This, combined with recent development within fluorescence imaging opens up for the use of encapsulated dyes for the <BR> <BR> detection of, e. g. , protein mediated ion translocation with high sensitivity and lateral resolution.

The length of the DNA label can be controlled, and thus used as a variable spacer, which is advantageous compared with previous vesicle-capture protocols, utilising avidin-mediated capturing of biotin-modified vesicles 1251, hydrophobic moieties in dextran gels'or immobilized antibodies \ One must be aware, though, that the substrates used here were kept hydrated throughout the series of modifications. There is no desire to circumvent the need for hydration in the final coupling of intact, functionalized vesicles.

However, recent progress demonstrating stabilised supported lipid bilayers that sustain drying without loosing their inertness towards protein adsorption, combined with robotic printing of DNA, or the use commercial DNA chips, points towards an expansion of the initial steps (i- iv in Fig. 1 and 2) of this protocol outside controlled laboratory conditions.

References [1] C. M. Niemeyer, D. Blohm, Angewandte Chemie-International Edition 1999,38, 2865.

[2] T. Kodadek, Chemistry & Biology 2001,8, 105.

[3] G. MacBeath, S. L. Schreiber, Science 2000,289, 1760.

[4] K. Glasmastar, C. Larsson, F. Hook, B. Kasemo, Journal of Colloid and Interface Science 2002,246, 40.

[5] D. Chapman, Langmuir 1993,9, 39.

[6] J. T. Groves, S. G. Boxer, Accounts of Chemical Research 2002,35, 149.

[7] W. Muller, H. Ringsdorf, E. Rump, X. Zhang, L. Angermaier, W. Knoll, J. Spinke, Journal of Biomaterials Science-Polymer Edition 1994,6, 481.

[8] F. Hook, A. Ray, B. Norden, B. Kasemo, Langmuir 2001,17, 8305.

[9] D. W. Pack, G. H. Chen, K. M. Maloney, C. T. Chen, F. H. Arnold, Journal of the American Chemical Society 1997,119, 2479.

[10] M. Egger, S. -P. Heyn, H. E. Gaub, Biochimica et Biophysica Acta 1992,1104, 45.

[11] H. B. Mao, T. L. Yang, P. S. Cremer, Analytical Chemistry 2002,74, 379.

[12] Y. Fang, A. G. Frutos, J. Lahiri, Journal of the American Chemical Society 2002,124, 2394.

[13] C. M. Niemeyer, Current Opinion in Chemical Biology 2000,4, 609.

[14] E. Soderlind, L. Strandberg, P. Jirholt, N. Kobayashi, V. Alexeiva, A.

M. Aberg, A. Nilsson, B. Jansson, M. Ohlin, C. Wingren, L. Danielsson, R.

Carlsson, C. A. K. Borrebaeck, Nature Biotechnology 2000,18, 852.

[15] P. S. Cremer, T. L. Yang, Journal of the American Chemical Society 1999,121, 8130.

[16] J. T. Groves, L. K. Mahal, C. R. Bertozzi, Langmuir 2001,17, 5129.

[17] L. A. Kung, L. Kam, J. S. Hovis, S. G. Boxer, Langmuir 2000,16, 6773.

[18] J. S. Hovis, S. G. Boxer, Langmuir 2001,17, 3400.

[19] S. Kunneke, A. Janshoff, Angewandte Chemie-International Edition 2001,41, 314.

[20] L. Kam, S. G. Boxer, Journal of the American Chemical Society 2000, 122,12901.

[21] J. F. Mooney, A. J. Hunt, J. R. McIntosh, C. A. Liberko, D. M. Walba, C. T. Rogers, Proceedings of the National Academy of Sciences of the United States of America 1996,93, 12287.

[22] R. Bashir, R. Gomez, A. Sarikaya, M. R. Ladisch, J. Sturgis, J. P.

Robinson, Biotechnology and Bioengineering 2001,73, 324.

[23] C. A. Keller, K. Glasmastar, V. P. Zhdanov, B. Kasemo, Physical Review Letters 2000,84, 5443.

[24] C. A. Keller, B. Kasemo, Biophysical Journal 1998,75, 1397.

[25] L. S. Jung, J. S. Shumaker-Parry, C. T. Campbell, S. S. Yee, M. H.

Gelb, Journal of the American Chemical Society 2000,122, 4177.

[26] M. A. Cooper, A. Hansson, S. Lofas, D. H. Williams, Analytical Biochemistry 2000,277, 196.

[27] C. R. MacKenzie, T. Hirama, K. K. Lee, E. Altman, N. M. Young, Journal of Biological Chemistry 1997,272, 5533.

[28] E. E. Ross, B. Bondurant, T. Spratt, J. C. Conboy, D. F. O'Brien, S. S.

Saavedra, Langmuir 2001, 17,2305.