Saposin Lipoprotein Particles and Libraries from Unpurified Membranes

Abstract

To provide a process for preparing a library of native state membrane proteomes and lipidomes in a form of saposin-derived particles.SOLUTION: Provided is a process for preparing a library of saposin lipoprotein particles where the library comprises a heterogenic mixture of saposin lipoprotein particles comprising different membrane lipids and optionally membrane protein compositions, where the process comprises the steps of: a) providing a mixture of crude membrane vesicles obtained from a cell or vesicle membrane of an archaeal, eukaryotic or prokaryotic organism, where the crude membrane vesicles contain both of the membrane lipids and membrane proteins from the source cell or vesicle membrane; b) contacting the mixture of step a) with the lipid binding polypeptide in a liquid environment; and c) allowing for self-assembly of the particles.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a process for preparing a library of lipoprotein particles from the membranes of cells or organelles and purifying the lipoprotein particles therefrom, the library of lipoprotein particles and the purified lipoprotein particles themselves, and their use.

Background Art

[0002] During the past decade, research in the life sciences, particularly in drug discovery, has been changing rapidly. Evolution in molecular biology, computing, genomics, proteomics and lipidomics has made it possible to develop new methods for research and drug discovery.

[0003] In this research area, libraries of the genomes, proteomes or lipidomes of cells or cell organelles of cells have been found to be very useful for studying the functions of genes, proteins or lipids and for discovering drug targets for genes, proteins or lipids. Although the fields of genomics and DNA libraries have also evolved sufficiently, there is still an urgent need to develop improved methods for preparing membrane proteome and / or lipidome libraries.

[0004] Drug discovery is based on the discovery, selection and further development of lead compounds having specific biological activities in a particular disease and / or, if a compound library is available, the identification of new drug targets in the cells involved in that disease. In many cases, the drug target is a lipid, lipid domain or membrane protein embedded in the membrane of a cell or organelle of the target cell, such as a receptor or transporter. Therefore, elucidation of the structure and function of lipids, lipid domains and membrane proteins embedded in their native membrane environment and the possibility of screening compounds or compound libraries against a complex library of possible lipid and membrane protein targets are generally highly desirable in life science research, particularly in the development of new pharmaceuticals.

[0005] For soluble proteins of cells or organelles, a library of soluble cell or organelle proteomes can be readily prepared by simply isolating the fraction of soluble proteins. Unfortunately, membranes (and the membrane proteins and lipids contained therein) are not soluble in detergent-free systems commonly used in most functional assays employed in drug screening procedures and life science research. Thus, current membrane proteome or lipidome libraries are mostly in a state dissolved or denatured in detergents. The latter may be useful for protein or lipid identification techniques such as mass spectrometry, but is incompatible with the functional analysis of membrane proteins or lipids in their native environment, which is a fundamental requirement for success in research and drug development in this field.

[0006] These difficulties in purification, isolation, and functional analysis have retarded the study of membrane lipidomes and membrane proteomes.

[0007] Lipidomics is the large-scale study of the pathways, composition, and networks of cellular lipids in biological systems, including the identification and quantification of the interactions of cellular lipid molecular species with other lipids, proteins, and other metabolites. The term "lipidome" is used to describe the totality of all lipids and / or lipid profiles of a cell, tissue, organism, or ecosystem, which is a subset of the "metabolome" that also includes other major classes of biomolecules such as proteins, amino acids, sugars, and nucleic acids. Lipidomics is a relatively recent field of study that has emerged through the rapid evolution of technologies such as mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy, fluorescence spectroscopy, and computer computational methods, in combination with the recognition of the role of lipids in many metabolic diseases such as obesity, atherosclerosis, stroke, hypertension, and diabetes. This rapidly expanding field complements the major advances made in genomics and proteomics, all of which constitute a group of systematic biology.

[0008] Lipidomes are very complex, and lipids play an essential role in membrane mechanics, energy metabolism, and signal transduction. The structure of lipids is a key determinant of biological effects. Analytical methods such as lipidomics are essential for enhancing the current biological understanding of biologically relevant lipids in basic research and pharmaceutical discovery and development. For this purpose, it is desirable to be able to provide a lipidome library in which lipids are maintained in a state as close as possible to their natural environment in the membranes of cells or organelles.

[0009] The techniques of lipidome research are applicable to all therapeutic areas, including cardiovascular diseases, diabetes, cancer, neurological diseases, autoimmune diseases, and inflammatory diseases. The basic requirements for successful lipidome research are sufficient pre-analytical samples, and preferably, a lipidome library that allows for rapid generation, easy handling, and short storage times, as certain lipids and naturally occurring lipid domains can be unstable.

[0010] Lipids also provide promising new biomarkers in many fields. In addition, they can also function as potential pharmacodynamic readouts for experimental or existing therapies. Therefore, lipids can drive the development of companion diagnostics and thus assist in more personalized treatment approaches. From this perspective, it is desirable to provide a simple and robust method for preparing a lipidome library. Lipidomics can also be used in the testing of various experimental disease models, which will lead to great progress in translational medicine.

[0011] On the one hand, proteomics is the large-scale study of proteins, particularly their structure and function. Proteins are a crucial part of living organisms as they are the main components of the physiological metabolic pathways and signal transduction pathways of cells. The proteome is the set of proteins made by and contained within an organism, cell, organelle, or system. The membrane proteome is the set of membranes and membrane-bound proteins made by and contained within the membrane of an organism, cell, organelle, or system. The proteome can vary over time in response to the state or stress that a cell or organism experiences, such as a disease.

[0012] The membrane proteome is a particularly interesting research target due to the many functions that membrane proteins perform in cell systems and diseases. Because of the abundance of potential drug targets, a library encompassing the membrane proteome of a given cell or organelle membrane would be highly desirable for the screening process. In addition to this, the isolation and identification of unknown membrane proteins provides new perspectives for discovering new pharmaceutical targets and identifying key biochemical receptors. As described above, the preparation and subsequent use of membrane proteome libraries has been hampered by the insolubility of membranes and membrane components in surfactant-free systems. The interaction between membrane protein targets and soluble ligands is difficult to test in vitro due to the inability of membrane proteins to dissolve in surfactant-free systems.

[0013] Hydrophobic compounds (e.g., membrane proteins and lipids) are notoriously difficult to handle and pose two main challenges for pharmaceutical research or life science research and applications. (i) Making insoluble hydrophobic compounds, such as lipids or membrane proteins, soluble in aqueous solutions, and (ii) handling and administering such hydrophobic substances as therapeutic, research, or diagnostic agents.

[0014] Membrane proteins are encoded by approximately 30% of all ORFs (Wallin and von Heijne, Protein Science 1998 Apr;7(4):1029-38) and represent an important class of drug targets because the majority of drugs (i.e., more than 60%) actually target this class of proteins (Overington et al., Nature Reviews Drug Discovery 5, 993-996 (December 2006)). Membrane proteins play important roles in many biological processes such as signal transduction, transport of molecules and energy, recognition, and cell-cell communication. However, membrane proteins are difficult to study because of their insolubility and tendency to aggregate when extracted from the native lipid bilayer environment. An artificial hydrophobic environment is required to maintain the integrity of membrane proteins. Here, surfactant micelles are most commonly used, but these can have an adverse effect on biocompatibility, can have an adverse effect on the activity of membrane proteins, and can interfere with the experimental conditions for assays.

[0015] Another major pharmacological challenge is represented by the administration and delivery of hydrophobic proteins and / or lipids as therapeutic and / or diagnostic agents. Due to the limited solubility of hydrophobic agents, these agents tend to aggregate, resulting in locally highly concentrated drug particles, which can cause high toxicity, unwanted immune responses, and can inactivate the drug (Allen and Cullis, SCIENCE, 303(5665):1818-1822, MAR 19, 2004).

[0016] Therefore, uses for incorporating hydrophobic agents such as membrane proteins or lipids into soluble particles are highly desired. Current methods for addressing these two challenges include, in particular, liposomes and reconstituted high density lipoprotein (rHDL) particles (Chan and Boxer, Current Opinion in chemical Biology 11:1-7, 2007).

[0017] European Patent No. 1596828 describes discoidal bioactive drug delivery particles containing apolipoproteins that closely surround a lipid bilayer in a double-belt-like fashion. The inside of these particles is created by the hydrophobic region of the lipid bilayer. This is in contrast to liposomes, which are closed spherical bilayer shells containing an aqueous interior. The discoidal bioactive drug delivery particles described in European Patent No. 1596828 have a Stokes diameter of about 10 nm and are proposed to be used as delivery vehicles for hydrophobic pharmaceutical drugs such as amphotericin B or camptothecin.

[0018] European Patent No. 1345959 describes similar types of nanoscale particles with a diameter of about 10 nm and a height of about 5.5 nm. These particles are discoidal and are composed of (i) an artificial membrane scaffold protein, (ii) a phospholipid bilayer, and (iii) at least one hydrophobic or partially hydrophobic protein. See Figure 1a below in this specification. This membrane scaffold protein also surrounds the lipid bilayer in a double-belt-like fashion and is a derivative of human apolipoprotein A-1 or a truncated form with the N-terminal globular domain of human apolipoprotein A-1 missing. It is amphiphilic, forms at least one α-helix, and self-assembles with phospholipids or a phospholipid mixture in an aqueous environment to form these discoidal nanoscale particles. Such engineered membrane scaffold proteins (MSPs) confer stability, size uniformity, and useful functions to nanoscale discoidal lipoprotein particles.

[0019] However, currently available nanodisc technology has several drawbacks, such as the need to remove surfactants, for example, in the particle assembly. Furthermore, the size uniformity provided by the tight double-belt-like fitting of MSPs derived from apolipoproteins seems to come at the expense of a fixed minimum particle size and a limited maximum diameter that can be obtained using prior art methods.

[0020] In recent years, novel nanoparticle technologies involving lipid-binding proteins of the saposin family have been proposed (see Qi et al. (2009) Clin Cancer Res 15(18):5840-5851, Popovic et al., PNAS, Vol. 109, No. 8 (2012) 2908-2912, International Publication No. 2014 / 095576 and International Publication No. 2015 / 036549).

[0021] The saposin-family includes four small (≈80 amino acids) proteins, saposins A–D, which bind to and / or interact with lipids and function as essential cofactors for some lysosomal enzymes in sphingolipid catabolism (see Bruhn, Biochem J. (2005) 389, 249-257 and references cited therein). Saposins have been described as preferring negatively charged lipids and low pH, showing significantly increased activity at acidic pH, with an optimal pH of 4.75 at the pH within liposomes. Saposins A, B, C and D are hydrolyzed by proteolysis from one large precursor protein, prosaposin. The complete amino acid sequences of saposins A, B, C and D have been reported together with the genomic constitution and cDNA sequences of prosaposin (O’Brien et al. (1988) Science 241, 1098-1101; Furst et al (1992) Biochim Biophys Acta 1126:1-16).

[0022] Saposin C can induce the membrane fusion of vesicles containing phospholipids in an acidic environment (Archives of Biochemistry and Biophysics 2003 Jul 1;415(1):43-53), and this feature is not shown by other saposins. Qi et al. (2009) Clin Cancer Res 15(18):5840-5851 reported saposin C-coupled dioleoylphosphatidylserine nanovesicles (SapC-DOPS) that have an aqueous interior, an average diameter of about 190 nm, and show tumor-targeting activity in vivo. In SapC-DOPS, saposin C or a peptide derived therefrom acts as a homing peptide for the liposomes to which it is attached. Saposin C then targets the liposomes to cancer cells with phosphatidylserine exposed on the outer leaflet of the cell membrane. These authors believe that the unique acidic microenvironment around cancer cells, which is due to the extracellular leakage of lysosomal enzymes, makes tumor tissue an optimal target for saposin C. According to Qi et al., SapC-DOPS liposomes are prepared by drying the purified phospholipids dissolved in a solvent under N2(g), dispersing the dried phospholipids in an acidic buffer (pH 5) containing purified saposin C, diluting this mixture 50-fold with a physiological aqueous solution, and facilitating the aggregation of nanovesicles by subsequent sonication.

[0023] Popovic et al., PNAS, Vol.109, No.8 (2012) 2908-2912 reported on the structure of saposin A surfactant discs. Saposin A exists in a state bound to soluble lipid / surfactant. In the absence of lipid, saposin A adopts a closed monomer apo conformation. In contrast, the saposin A surfactant disc structure reported by Popovic et al. involves 40 surfactant molecules bound to the inside, organized in a bilayer-like hydrophobic core in which two chains of saposin A in an open conformation are highly regularly arranged and encapsulated.

[0024] In addition to the crystallization of the saposin A surfactant disk, Popovic et al. also describe the preparation of soluble lipid-saposin A complexes at pH 4.75 by a method that requires multiple steps. First, the purified lipid dissolved in chloroform is dried under N2(g), and the dried lipid is dispersed in an acidic buffer (50 mM sodium acetate pH 4.8, 150 mM NaCl) by vortex mixing. For the suspension, 10 cycles of freeze-thaw are performed, blended for 5 minutes with a vortex mixer, and the mixture is extruded through a 200 nm filter to prepare a homogeneous fraction of large unilamellar liposome vesicles. When the large unilamellar artificial liposome vesicles thus prepared are mixed with purified saposin A in an acidic buffer, soluble lipid-saposin A particles are obtained. These particles showed a narrow particle size distribution around an average hydrodynamic (Stokes) radius of 3.2 nm and contained approximately 5:1 lipid molecules per saposin A chain. The actual size of these particles was only moderately affected by the molar ratio of lipid to protein and the composition of the liposome. The authors observed that similar 3.2 nm particles were present in the liposome mixture regardless of whether the anionic phospholipid, cholesterol, or glycosphingolipid was used. In all cases, one peak was observed in the particle size range of Stokes radius 3.2 nm, indicating that this species has a relatively narrow particle size distribution. Thus, the technique of this publication is limited to a pH value of 4.75, limited to particles of the above-described particle size, and includes a cumbersome upstream liposome preparation process.

[0025] WO 2014 / 095576 was the first to show that a hydrophobic cargo molecule solubilized in a surfactant or a membrane protein purified and solubilized in a surfactant can be incorporated into saposin-lipid particles (see Figure 1b below in this specification). Thus, the method described in WO 2014 / 095576 uses components solubilized in a purified surfactant and compares this to synthetic liposomes prepared from lipids purified by the method of Popovic et al.

[0026] International Publication No. WO 2015 / 036549 extended the method described in International Publication No. WO 2014 / 095576 to the incorporation of solubilized antigen molecules (shown for viral membrane proteins) from well-defined, purified HIV-1 virus-like particles (VLPs). According to the examples of International Publication No. WO 2015 / 036549, pre-purified VLPs are lysed, HIV-1 membrane spike proteins are solubilized with a surfactant, and then contacted with saposin A. In general terms, International Publication No. WO 2015 / 036549 also suggests that solubilized antigen molecules from microorganisms, fungi, protozoa, parasites or human or animal neoplasms / tumors are in principle usable, but no experimental details are given. Also, only purified components are used in a solubilized state with a surfactant, i.e., the native membrane content is not maintained.

[0027] A wide variety of hydrophobic agents (e.g., membrane proteins or lipids) would benefit from the apolipoprotein-derived or saposin-derived nanodisc technology described in the prior art. It can be readily imagined that the preparation of lipidosomes and membrane proteome libraries requires that the nanodisc particles be very flexible in terms of size and cargo compatibility. However, due to the size of the saposin A-derived particles reported by Popovic et al. being limited to 3.2 nm, it would seem that only low molecular weight molecules can be incorporated into such particles at the disclosed acidic pH, if at all. Bulky hydrophobic compounds and large biomolecules, e.g., (oligomeric) membrane proteins, can be incorporated into nanodiscs derived from apolipoprotein A of the prior art, but the maximum possible diameter is still limited by the double-belt-like apolipoprotein A around these particles. In addition, nanodiscs derived from 10 nm apolipoprotein A may be too large for certain applications.

[0028] Furthermore, all of the prior art methods described for apolipoproteins or saposins are precise at the level of experimental detail, purified, and rely on a well-defined system of components solubilized in detergents. Thus, these methods are expected not to work when starting directly with unpurified membranes characterized by highly complex structures and compositions. If the first is for purifying and solubilizing membrane lipidomes and proteomes from the membranes of cells or organelles, however, it removes membrane proteins and lipids from their native environment and contents, and in the library composition, the function is lost, and the contents, complexity, and corresponding bias are lost. In addition to this, such processes will become complex and elaborate because not all membrane proteins and membrane lipids have the same requirements with respect to handling, detergent solubilization, and stability.

[0029] Also, the above-described prior art processes showing the incorporation of bacterial or eukaryotic membrane proteins use synthetic or purified lipids to reconstitute lipoprotein particles. Thus, the lipids and membrane proteins present in the lipoprotein particles are from different sources and do not reflect the naturally occurring environment. However, mimicking such a naturally occurring environment is highly desirable for obtaining a library that gives meaningful results in research and screening, increases the possibility of being mobile and confirmable in native cells, and, if possible, in the contents of diseases.

[0030] In summary, most of the prior art methods used to incorporate prokaryotic or eukaryotic membrane components (such as proteins and lipids) into nanoparticle structures involve, in particular, when using synthetic lipids or lipids purified from completely different sources, the extraction, solubilization, and reassembly of the components into the particles. These particles do not resemble the naturally occurring membranes that are their source. This makes it impossible to observe the naturally occurring membrane environment. In addition, these known procedures carry the risk of protein denaturation or destabilization of lipids and lipid domains during purification and extraction. Furthermore, it cannot be excluded that proteins and / or lipids loosen their native structure and function when removed from their native environment.

[0031] The prior art related to saposins teaches only the incorporation of membrane proteins purified by and / or solubilized with surfactants from prokaryotic or eukaryotic sources, or solubilized antigens from purified artificial virus-like particles. These techniques give a very homogeneous collection of nanodiscs containing the purified membrane proteins of interest reassembled in a well-defined lipid environment, but are usually quite different from the naturally occurring membranes of prokaryotic, archaeal, and eukaryotic organisms.

[0032] In contrast, these naturally occurring membranes of prokaryotic, archaeal, and eukaryotic organisms are very complex, containing a very diverse array of interacting proteins and lipids, and form complex superstructures with the ability to control various cellular processes. Without performing the surfactant solubilization and purification steps taught by prior art methods, the handling and incorporation of such complex membranes seems almost impossible because the complex native membrane structures are not expected to be as easy to handle as the purified components. In particular, natural membranes consist mainly of different types of phospholipids, including complex mixtures and domains of various lipid components containing POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine), and sphingomyelin, and other lipids such as cholesterol. Furthermore, the membrane may form domains (such as lipid rafts) that contain a significantly higher proportion of cholesterol and sphingomyelin than, for example, the general plasma membrane that aids protein signaling. A given membrane protein may function properly only within the native membrane context.

[0033] Taken together, the membranes of prokaryotic, archaeal, and eukaryotic organisms exhibit a high degree of complexity arising from the complex interactions between membrane proteins and the various lipids surrounding them in the native context.

[0034] The prior art does not envision, and certainly does not teach or suggest, a feasible proposal for preparing libraries of native state membrane proteomes and lipidomes in the form of saposin-derived particles.

[0035] However, as described above, there is a great need for libraries in which membrane proteome or lipidome components remain preserved in their native environment. For example, elucidating the structure, function, and interactions of membrane proteins in vitro in their native environment, i.e., membrane proteins that exist in vivo and are embedded in the active membrane lipids, would be useful for identifying drug targets and understanding the underlying mechanisms and reactions that occur at the level of the membranes of cells and organelles. Therefore, there is a need to develop a novel method for preparing libraries from the membranes of cells or organelles that can preserve membrane lipids and membrane proteins in their native environment so as to maintain their respective structures and functions. By providing a library that reflects the lipid and protein composition of the unpurified membrane, it will be possible to test the lipidome, proteome, or membrane structure of a particular cell, organelle, or organism at a level that has not been achievable heretofore.

Summary of the Invention

[0036] In view of this background, the problem underlying the present invention will be understood by the provision of improved lipoparticle proteins, a library of improved lipoparticle proteins, and methods for producing these.

[0037] This problem is solved by the process of the present invention that uses unpurified membrane vesicles as starting material for preparing a library of lipoparticle proteins comprising a lipid-binding polypeptide and at least a part or component of a cell or organelle membrane, wherein the lipid-binding polypeptide is a saposin-like protein or a derivative thereof or a form with a truncated tip.

[0038] The present invention is a process for preparing a library of saposin lipoparticle proteins, wherein the particles comprise a membrane component from a cell or organelle membrane and a lipid-binding polypeptide that is a saposin-like protein belonging to the SAPLIP family of proteins that interact with lipids or a derivative form thereof, and the process comprises a) providing a mixture of unpurified membrane vesicles obtained from cell or organelle membranes; b) contacting the mixture of step a) with the lipid-binding polypeptide in a liquid environment; c) self-assembling the particles, to provide a process.

[0039] The process of the present invention can be extended to a purification process for specific types of saposin lipoproteins particles, the process comprising preparing a library according to the process described above, f) further purifying at least one type of saposin lipoprotein particle from the library.

[0040] The saposin lipoprotein particles described herein contain membrane components (especially membrane lipids) and optionally also membrane proteins, all derived from cell or organelle membranes. The saposin lipoprotein particles described herein further contain a saposin-like protein (SAPLIP) belonging to the SAPLIP family of proteins that interact with lipids or a lipid-binding polypeptide in its derivative form, which can be obtained by the method of the present invention. These saposin lipoprotein particles described herein are also referred to herein as "Salipro particles", "saposin lipoprotein particles" or "particles of the present invention". A library containing a mixture of different Salipro particles, which can be obtained by the method of the present invention, is also referred to herein as a "Salipro particle library".

[0041] The process according to the present invention is relatively easy to carry out, reflects the diversity and complexity differences of the unpurified membranes used as starting materials, incorporates and preserves the lipid and membrane protein components contained in their native membrane environment, and has the advantage that a library of sariplo particles can be obtained. A wide variety of membranes may be used in the process according to the present invention. The unpurified membrane vesicles used in step a) are prepared from the unpurified membranes of cells or organelles. Unpurified membrane vesicles obtained from the membranes of cells and / or organelles of many organisms may be used. Surprisingly, even when using the complex membranes of eukaryotic, prokaryotic or archaeal cells as starting materials instead of the highly purified and surfactant-solubilized starting materials required by prior art preparation methods, sariplo particles could be obtained.

[0042] The sariplo particles present in the library obtained using the process according to the present invention vary in size and composition, contain different mixtures of membrane components, and reflect the membrane lipidome and proteome of the unpurified membrane of the starting material. The components of the unpurified membranes obtained from the organelles of cells may also be incorporated into the sariplo particles.

[0043] Those skilled in the art know that the membranes obtained from cells and organelles exhibit a complex and highly heterogeneous mixture of components, and these components are expected to interact with lipid-binding polypeptides and other reagents used in the process of preparing lipoprotein particles. Thus, it was expected that little product would be obtained, or that a low-quality product or a poorly defined product would be obtained. Against this background, it was surprising that complex unpurified membranes could be used and an array of their diverse components could be easily incorporated into sariplo particles to create the corresponding membrane proteome / lipidome library. In contrast to the process according to the present invention that uses unpurified membrane vesicles, prior art processes that use saposin-like proteins together with eukaryotic or prokaryotic membrane proteins mainly reconstitute with highly purified proteins or use lipids or synthetic lipids that are purified such that controlled conditions are provided.

[0044] The process according to the present invention provides a library that efficiently captures membrane components, particularly membrane lipids and membrane proteins, directly from complex cell membranes. Since the process according to the present invention uses unpurified membrane vesicles (i.e., intact physiological membrane portions and / or components that have not been extracted or purified as in the prior art methods) and includes both their various native lipids and their native membrane proteins, the library of sariplo particles obtained by the process according to the present invention contains a complex array of membrane components. This makes it possible to provide a library representing the lipidome and / or membrane proteome from a specific cell or organelle.

[0045] In particular, the process according to the present invention enables the preparation of a library containing a heterogeneous mixture of sariplo particles comprising different membrane lipids and optionally a membrane protein composition. As described above, providing such a library containing an array of membrane components of a heterogeneous array, i.e., an array of sariplo particles composed of membrane lipids and / or membrane proteins embedded in their native environment, is a very useful tool in drug discovery, antibody generation, membrane (protein or lipid) research, lipidomics, proteomics or medical, cosmetic and diagnostic applications.

[0046] From actual experiments, it has been found that the size of sariplo particles is self-adjusted with respect to the nature of the membrane components incorporated, for example, with respect to the size of the membrane proteins incorporated. Sariplo particles are surprisingly flexible in size and thus allow for further flexibility and / or variation in the specificity of different lipids (and optionally membranes) in a library-like membrane lipidome or proteome compared to other scaffold proteins and agents used in today's membrane library technologies.

[0047] While not bound by theory, the process according to the invention appears to enable the saposin particles to adjust their size with respect to the properties of the membrane components to be incorporated. This is advantageous as the size of other conventional particles is restricted. This flexibility also clearly enables the incorporation of membrane proteins into their native environment (e.g., membrane lipids or other cellular components potentially required to associate with membrane proteins and maintain the protein's structure and / or function). It is also surprising that such complex membranes and components of varying size and composition are successfully incorporated into saposin particles according to the process of the invention, and that the resulting library is nevertheless stable and can be processed, handled, purified, and / or analyzed without further difficulty. The saposin particles obtained using the process according to the invention are easy to manufacture and can maintain a uniform quality and composition over time, thereby offering the possibility of providing a stable and valuable library for further processes and applications.

[0048] While not bound by theory, when in step b) of the method according to the invention, unpurified membrane vesicles are contacted with a saposin-like protein or a derivative thereof or a truncated form, and self-assembled in step c), a robust structure is obtained that is stable over a wide pH range, particularly in aqueous solution at physiological pH, and appears to be capable of forming particles larger than the 3.2 nm lipo-protein particles derived from saposin A synthesized according to the prior art teachings of Popovic et al.

[0049] As described in the introduction, the importance of membrane proteins in therapeutic drug development requires the discovery of innovative methods for examining membrane proteins in cell-free media, preferably in a surfactant-free environment.

[0050] The libraries and suripro particles obtainable by the method of the present invention meet this need. Once the libraries and suripro particles are obtained, they are stable in a cell-free medium and a surfactant-free environment. In one embodiment, the use of a surfactant is not essential in this process.

[0051] The crude membrane contains a large number of different membrane proteins and lipid components. In contrast to what would be expected, when no surfactant is present, the membrane proteins are insoluble in a surfactant-free buffer system and aggregate, generating large invalid peaks in SEC analysis. In actual experiments, it was found that when encapsulated in suripro particles, the membrane proteins and membrane lipid components remain soluble in a surfactant-free buffer system.

[0052] In another aspect of the present invention, a library of suripro particles may be used to further purify specific types of suripro particles, i.e., suripro particles containing a specific membrane protein or lipid composition of interest.

[0053] Furthermore, the libraries and particles obtainable by the method of the present invention can be used as tools for drug development, drug screening, drug discovery, antibody development, development of therapeutic biopharmaceuticals, for membrane or membrane protein purification, for membrane protein expression, for membrane and / or membrane protein research, particularly for lipidomics and proteomics, preferably for the isolation, identification and / or testing of membranes and / or membrane proteins, or for the creation of lipidome or proteome databases.

[0054] Finally, a library of suripro particles or suripro particles may be used in medicine, particularly for use in preventing, treating or reducing the severity of a disease, or for use in diagnostic methods, cosmetic treatments, or as a vaccine formulation. Detailed Description of the Invention

[0055] The present invention is a process for preparing a library of saposin-lipoprotein particles, said particles comprising membrane components from cells or organelle membranes and a lipid-binding polypeptide which is a saposin-like protein belonging to the SAPLIP family of proteins that interact with lipids or a derivative form thereof, said process comprising a) providing unpurified membrane vesicles obtained from cells or organelle membranes; b) contacting the mixture obtained from step a) with said lipid-binding polypeptide in a liquid environment; c) self-assembling said particles.

[0056] The process according to the present invention provides in particular a library of sariplo particles, each sariplo particle comprising a lipid-binding polypeptide, membrane lipids and optionally membrane proteins. The term "membrane protein" as used herein does not include the lipid-binding polypeptides of the present invention. "Membrane lipids" as used herein is a mixture of membrane lipids, in particular a naturally occurring mixture of membrane lipids. "Membrane lipids" as used herein is derived from the unpurified cells or organelle membranes from which the unpurified membrane vesicles are prepared. Thus, membrane lipids as used herein do not include pre-purified lipids or lipid mixtures.

[0057] "Unpurified cell or organelle membranes", as used herein, are membranes of cells or organelles, or portions thereof, that are no longer completely intact but still essentially contain the composition of natural membranes, particularly with respect to membrane lipids and membrane proteins. For example, an unpurified membrane fraction obtained from cell disruption or lysis of cells or organelles is an "unpurified cell or organelle membrane" according to the present invention. "Unpurified cell or organelle membranes" necessarily contain the natural membrane components present in cells and organelles. In particular, "unpurified cell or organelle membranes" contain both membrane lipids and membrane proteins. In a preferred embodiment, the membrane lipids and membrane proteins present in the sariplo particles of the present invention, or the membrane lipids and membrane proteins present in the sariplo particle library of the present invention, are obtained from the membranes of the same cells and / or cell organelles.

[0058] Since the unpurified cell or organelle membranes are no longer completely intact cell or organelle membranes, unpurified membrane vesicles naturally form due to hydrophobic interactions between two given membrane disruption sites. Thus, in one embodiment, "unpurified cell or organelle membranes" includes "unpurified membrane vesicles", or the two terms are used synonymously.

[0059] The term "library" as used in the present invention means a series of (complex plural) different sariplo particles. In particular, this difference will be in the size and composition of the particles, particularly the composition of the membrane components (i.e., membrane lipids and optionally membrane proteins) contained in the particles. Typically, the library is a mixture of "lipid-only particles" (see Figures 2a and 2b) and sariplo particles containing different types of membrane proteins (see Figures 2c to 2f). The particles in the library may differ in terms of their different membrane lipid contents and compositions. Preferably, some of the particles in the library differ in terms of whether the particle contains a membrane protein and which particles contain a membrane protein.

[0060] The membrane lipids and / or membrane proteins in the sapro particles are obtained from the membrane of a specific cell, all the membranes of a specific cell, the membrane of a specific organelle, all the membranes of a specific organelle, all the membranes of a specific individual or organism, or any other possible membrane sample containing membranes from cells or cell organelles of cells. Preferably, the sapro particles included in the library differ in terms of the composition of their membrane lipids and / or membrane proteins. Preferably, they differ in terms of the composition of their membrane proteins.

[0061] In a preferred embodiment, the sapro particles are disk-shaped. In another preferred embodiment, the sapro particles do not contain an aqueous or hydrophilic core. In yet another embodiment, the sapro particles are disk-shaped and do not contain an aqueous or hydrophilic core.

[0062] Accordingly, the present invention is a process for preparing a library of discoidal saposin lipoprotein particles, wherein said particles comprise a membrane lipid and a lipid-binding polypeptide which is a saposin-like protein belonging to the SAPLIP family of proteins that interact with lipids or a derivative form thereof, said particles do not contain a hydrophilic or aqueous core, and said process comprises a) providing unpurified membrane vesicles obtained from cell or cell organelle membranes; b) contacting the mixture obtained from step a) with said lipid-binding polypeptide in a liquid environment; c) self-assembling said particles, particularly preferably at a pH of 2.0 to 10.0, particularly 6.0 to 10.0, preferably 6.0 to 9.0, particularly preferably 7.0 to 9.0, most preferably 7.0 to 8.0. The present invention also provides a process comprising these steps.

[0063] The particles of the present invention have been found to be able to incorporate various membrane lipids and optionally membrane proteins, are soluble in an aqueous environment, and form stable nanoscale complexes. In particular, the particles according to the present invention are nanoscale particles comprising a lipid-bound polypeptide, a membrane lipid, and optionally a membrane protein. The membrane lipid and optionally the membrane protein are preferably obtained from the membranes of the same cell and / or organelle, and in particular, from the membranes of the same plurality of cells and / or organelles.

[0064] In a preferred embodiment, the particles of the present invention are generally considered to be disk-shaped. In particular, these particles may have a Stokes radius (hydrodynamic radius) RS in the range of 2 nm to 200 nm, particularly 3 nm to 150 nm, preferably 3 nm to 100 nm. Those skilled in the art know how to determine the Stokes radius. This is preferably done by analytical gel filtration (size exclusion chromatography) compared to the standard of the standard Stokes radius. In particular, for the particles, for example, a gel filtration step may be performed on a Superdex 200 HR10 30 gel filtration column and eluted at room temperature at 0.5 ml / min with an appropriate buffer at pH 7.5. The absorbance is monitored at 280 nm for proteins. The column is calibrated using a mixture of protein standards having known Stokes radii such as thyroglobulin 669 kDa (RS = 8.5 nm), ferritin 440 kDa (RS = 6.1 nm), catalase 232 kDa (RS = 4.6 nm), lactate dehydrogenase 140 kDa (RS = 4.1 nm), bovine serum albumin 66 kDa (RS = 3.55 nm), and horse heart cytochrome c 12.4 kDa (RS = 1.8 nm). The standard proteins should have RS values that span above and below the Rs value of the particle of interest. A calibration curve is made by plotting the elution position against the RS of the standard proteins. This generally gives a nearly linear plot, but otherwise, it is advisable to draw a line between the points and the reading of the Rs of the protein of interest from its elution position on this standard curve.

[0065] In some embodiments, for example, when bulky hydrophobic agents, such as membrane proteins, or large amounts of lipids are present in the particles, the Stokes radius will be greater than 3.2 nm, and in particular, at least 3.5 nm, at least 5.0 nm or at least 10.0 nm.

[0066] The particles of the present invention may be tested by a transmission electron microscope, or, if the particles are large enough, may be tested by negative staining electron microscopy and single-particle analysis.

[0067] Structural analysis has in many cases shown that in the particles of the present invention, the membrane lipids assemble into a discoidal bilayer-like structure having distinct sizes inside the particles (see FIGS. 2a and 2b). The lipid-binding polypeptide component generally defines the boundary of the discoidal bilayer and gives structure and stability to the particles. In most embodiments, the interior of the particles contains a hydrophobic region (e.g., composed of lipid aliphatic acyl chains). In contrast to liposomes, the particles of the present invention preferably do not contain a hydrophilic or aqueous core. The particles preferably have a discoidal shape and have a flat, substantially circular lipid bilayer, and are surrounded by amphiphilic α-helices of two or more lipid-binding polypeptides, and are associated with the hydrophobic surface of the bilayer around the disc. Specific examples of the discoidal-shaped particles of the present invention are schematically shown in FIGS. 2a-2f.

[0068] In some embodiments, the disk shape of the particles of the present invention will be approximated by a cylindrical shape with a ratio of maximum height to maximum diameter (length of the major axis) of at least 1.0:1.1, particularly 1.0:1.5 or 1.0:2.0. The maximum height of the disk-shaped particles is generally at least 3.5 nm, particularly at least 5 nm, when determined by transmission electron microscopy or, if the particles are large enough, by negative staining electron microscopy and single-particle analysis. Preferably, the particles of the present invention have an upper surface, a bottom surface, and a surrounding side surface, and the maximum diameter (length of the major axis) of the upper and bottom surfaces is greater than the height of the surrounding side surface. In some embodiments of the particles of the present invention, the lipid-conjugated polypeptide is at least partially disposed around the surrounding side surface of the particles.

[0069] In some embodiments of the present invention, the maximum diameter (length of the major axis) of the disk-shaped particles of the present invention is 2 nm to 200 nm, particularly 3 nm to 150 nm, preferably 3 nm to 100 nm, when determined by transmission electron microscopy or, if the particles are large enough, by negative staining electron microscopy and single-particle analysis. In another embodiment, the maximum diameter (length of the major axis) of the disk-shaped particles is 3 nm to 80 nm, particularly 3 nm to 60 nm. Actual experiments have shown that particles with a maximum diameter (length of the major axis) of 3 nm to 20 nm can be obtained particularly easily using the method of the present invention.

[0070] In a preferred embodiment of the present invention, the particles are defined by a substantially monodisperse collection of disk structures, for example, when evaluated by a gel filtration elution profile on a HiLoad Superdex™ 200 16 / 60 GL column.

[0071] Generally, the preferential interaction between the lipid-bound polypeptide and the lipid bilayer within the particle is due to hydrophobic interactions between residues on the hydrophobic surface of the amphipathic α-helix of the lipid-bound polypeptide molecule and the hydrophobic surface of the lipid (e.g., phospholipid aliphatic acyl chains) at the edge of the bilayer surrounding the bioactive agent delivery particle. The amphipathic α-helix of the lipid-bound polypeptide molecule has a hydrophobic surface that contacts the hydrophobic surface of the lipid bilayer surrounding the particle and a hydrophilic surface that faces outside the particle and contacts the aqueous environment when the particle is suspended in an aqueous medium.

[0072] In some embodiments, the libraries and particles according to the invention are stable in aqueous solution and may be lyophilized for long-term storage and subsequent reconstitution in aqueous solution. "Stability" or "stable", as used herein, means that during the preparation, transport and storage of the particles, particle fragmentation is low to undetectable levels and aggregation or quality degradation is low to undetectable levels.

[0073] In a preferred embodiment, the library and particles according to the present invention are stable in an aqueous solution at a pH of 2.0 to 10.0, particularly 6.0 to 10.0, preferably 6.0 to 9.0, particularly preferably 7.0 to 9.0, and most preferably 7.0 to 8.0. In another embodiment, the library and particles according to the present invention are, for example, when determined by visual observation (a clear solution without precipitation) or by analytical gel filtration (fragmentation of less than 50%, particularly 1 to 40% of the particles), stable in an aqueous solution at a temperature of -210°C to 80°C, particularly -210°C to 40°C, -210°C to 30°C or -210°C to 4°C for at least 1 day, at least 2 days, at least 7 days, at least 2 weeks, at least 1 month, at least 6 months or at least 12 months. Actual experiments have shown that the particles of the present invention are stable in an aqueous solution at a pH of 5.0 to 8.0 and a temperature of 4°C to 40°C for at least 1 day, at least 2 days, at least 7 days, at least 2 weeks, at least 1 month or at least 3 months, for example, when determined by visual observation (a clear solution without precipitation) or by analytical gel filtration (fragmentation of less than 50%, particularly 1 to 40% of the particles). The particles of the present invention are known to be stable in an aqueous solution at a pH of 5.0 to 8.0 and a temperature of 40°C to 75°C for at least 10 minutes, for example, when determined by visual observation (a clear solution without precipitation) or by analytical gel filtration (fragmentation of less than 50%, particularly 1 to 40% of the particles). In some embodiments, the particles may be lyophilized for long-term storage and subsequent reconstitution in an aqueous solution. In some embodiments, the particles of the present invention are stable in the lyophilized form at a temperature of -210°C to 80°C, particularly -210°C to 40°C, -210°C to 30°C or -210°C to 4°C for at least 1 day, at least 2 days, at least 7 days, at least 2 weeks, at least 1 month, at least 6 months or at least 12 months, for example, when determined by analytical gel filtration (fragmentation of less than 50%, particularly less than 40%, or 1 to 40% of the particles) after reconstitution at pH 7.5 with an appropriate buffer."Fragmentation", as used herein, means that in the gel filtration elution profile, the size (i.e., peak height) of the peak corresponding to the particles of the present invention is reduced due to the peak size of free SAPLIP and / or free lipid and / or aggregates not bound to lipid, as compared to the size of the peak of the freshly prepared particles of the present invention. Thus, 40% fragmentation means, for example, that the size of the peak (i.e., the height of the peak in the gel filtration elution profile) is reduced by 40% compared to the size of the peak before storage (100%).

[0074] From actual experiments, it has been shown that the particles of the present invention are stable, especially in an aqueous solution substantially free of surfactant. Substantially free of surfactant means that the aqueous solution contains less than 0.001% (w / v) surfactant based on the total volume of the aqueous solution.

[0075] The lipid-binding polypeptide used in accordance with the present invention, i.e., the lipid-binding polypeptide in the saposin-like particle, is a saposin-like protein (SAPLIP), or a derivative or truncated form thereof. The term "saposin-like protein" (SAPLIP) as used herein is recognized in the art and includes all members of the saposin-like protein (SAPLIP) family of proteins that interact with lipids. The SAPLIP family is characterized by a saposin-fold moiety and a conserved α-helical three-dimensional structure stabilized by highly conserved intramolecular disulfide bonds (Munford et al. (1995), Journal of Lipid Research, vol. 36, no. 8, 1653-1663 and Bruhn (2005), Biochem J 389(15):249-257). Examples of the saposin-like protein (SAPLIP) family according to the present invention are described in Munford et al. (1995), Journal of Lipid Research, vol. 36, no. 8, 1653-1663 and Bruhn (2005), Biochem J 389(15):249-257, both of which are hereby incorporated by reference in their entirety.

[0076] In the ligand-free (i.e., surfactant-free / lipid-free) "closed" state, SAPLIP forms the saposin-folded portion where four helices of the monomeric compression type are bundled. This folded portion is exemplified by the structure of the human saposin A closed apo form (Protein Data Bank (PDB) ID code: 2DOB, Ahn et al. (2006) Protein Sci. 15:1849-1857), or the structure of saposin C (PDB ID code: 1M12; de Alba et al. (2003) Biochemistry 42, 14729-14740), NK-lysin (PDB ID code: 1NKL; Liepinsh et al. (1997) Nat. Struct. Biol. 4, 793-795), amoeba pore A (PDB ID code: 1OF9) and granulysin (PDB ID code: 1L9L; Anderson et al. (2003) J. Mol. Biol. 325, 355-365), all of which are nearly identical and can be easily superimposed.

[0077] When SAPLIP binds to a ligand, such as a lipid or a surfactant molecule, it undergoes a conformational change. In the "open" conformation with the ligand bound, SAPLIP adopts a V-shaped or boomerang-shaped conformation, with a hydrophobic surface exposed that contacts the bound lipid. This open conformation is exemplified by the prior art saposin A surfactant disk structure (PDB ID code: 4DDJ; Popovic et al., PNAS, Vol. 109, No. 8 (2012) 2908-2912) and the structure of saposin C bound to an SDS surfactant micelle (PDB ID code: 1SN6; Hawkins et al. (2005) J. Mol. Biol. 346:1381-1392).

[0078] In the particles of the present invention, the lipid-binding polypeptide is preferably amphiphilic, a part of its structure is hydrophilic to some extent and faces an aqueous solvent, and the other part is hydrophobic to some extent and faces the hydrophobic core of the lipid-containing particle. The lipid-binding polypeptide preferably has a more hydrophobic residue (e.g., A, C, F, G, I, L, M, V, W or Y) preferentially on one side of the helix and a more polar or charged residue (e.g., D, E, N, Q, S, T, H, K or R) on the other side of the helix, and is characterized by an amphiphilic α-helix.

[0079] The abbreviations of amino acid residues, when used herein, are as follows. A, Ala, alanine; V, Val, valine; L, Leu, leucine; I, Ile, isoleucine; P, Pro, proline; F, Phe, phenylalanine; W, Trp, tryptophan; M, Met, methionine; G, Gly, glycine; S, Ser, serine; T, Thr, threonine; C, Cys, cysteine; Y, Tyr, tyrosine; N, Asn, asparagine; Q, Gln, glutamine; D, Asp, aspartic acid; E, Glu, glutamic acid; K, Lys, lysine; R, Arg, arginine; and H, His, histidine.

[0080] In contrast to nanodiscs derived from prior art apolipoproteins, the lipid-binding polypeptides of the present invention do not have lipids surrounded in a double-belt-like fashion. Rather, the particles of the present invention are held together by a core containing lipids surrounded by two or more lipid-binding polypeptides in a head-to-tail arrangement in a substantially V-shaped or boomerang-shaped configuration, and there is substantially no direct protein-protein contact between the individual lipid-binding polypeptides within a given particle of the present invention (see FIGS. 1a to 1f). Without wishing to be bound by this theory, this arrangement of lipid-binding polypeptides and lipids in the particles of the present invention is thought to give the degree of freedom in size observed when a bulky hydrophobic agent is incorporated into the particles of the present invention or the amount of lipid incorporated increases.

[0081] The ability to interact with lipids, and the amphiphilicity described above, and the three-dimensional structure are highly conserved among SAPLIPs, but these are very diverse at the amino acid sequence level, and the sequence identity is lower than the normal threshold region of 25-30% identity that defines homology (see the sequence comparisons in FIGS. 4A and 4B of Bruhn (2005), Biochem J 389(15):249-257, reproduced in FIGS. 12a and 12b below, the sequences shown here form part of the disclosure of the present invention).

[0082] In the lipoprotein particles of the present invention, the lipid-binding polypeptide mainly serves as a structural protein and provides a scaffold for the structure of the lipoprotein particles of the present invention, for example, a discoidal structure. For this reason, structural features, in particular, the saposin-fold portion characteristic of SAPLIPs, are more important for defining the lipid-binding polypeptide of the present invention compared to mere sequence determinants.

[0083] Examples of SAPLIPs according to the present invention are saposin A, B, C or D (e.g., from Homo sapiens [see SEQ ID NOs: 1-4], Equus caballus, Bos taurus, Mus musculus, Oryctolagus cuniculus, Rattus norvegicus or Xenopus laevis); surfactant protein B (e.g., from Homo sapiens, Canis familiaris, Mus musculus, Oryctolagus cuniculus, Ovis aries or Rattus norvegicus); granulysin (e.g., from Homo sapiens; see SEQ ID NO: 5); NK-lysin (e.g., from Sus scrofa; see SEQ ID NO: 6); NK-lysin ortholog (e.g., from Equus caballus or Bos taurus); amoebapore (e.g., from Entamoeba histolytica); amoebapore ortholog (e.g., from Entamoeba dispar or Entamoeba invadens); amoebapore-like protein (e.g., from Fasciola hepatica); Naegleria pores (e.g., from Naegleria fowleri); Clornorin (e.g., from Clonorchis sinensis); prosaposin (e.g., from Homo sapiens, Equus caballus, Bos taurus, Mus musculus, Oryctolagus cuniculus, Rattus norvegicus or Xenopus laevis) and MSAP (e.g., from Homo sapiens).

[0084] The sequences of specific SAPLIPs used in accordance with the present invention are given in FIGS. 4A and 4B of Bruhn (2005), Biochem J 389(15):249-257, and the figures and sequences specified herein form part of the disclosure of the present invention and are thus reproduced again in FIGS. 12a and 12b below. The sequences of specific SAPLIPs used in accordance with the present invention are given in the following sequence listing. SEQ ID NO: 1, Sapocin A [Homo sapiens]; SEQ ID NO: 2, Sapocin B [Homo sapiens]; SEQ ID NO: 3, Sapocin C [Homo sapiens]; SEQ ID NO: 4, Sapocin D [Homo sapiens]; SEQ ID NO: 5, Granulysin [Homo sapiens]; SEQ ID NO: 6, NK-lysin [Sus scrofa].

[0085] The SAPLIPs used in accordance with the present invention may be polypeptides that include a sapocin-fold portion as part of a multi-domain protein. This may apply, for example, to acid sphingomyelinase (from Homo sapiens, Caenorhabditis elegans, Ciona intestinalis, Anopheles, Drosophila, Mus musculus or Rattus norvegicus); GDSL (Gly-Asp-Ser-Leu) lipases, such as acyl-oxy hydrolase (from Homo sapiens or Rattus norvegicus); Countin (from Dictyostelium discoideum); J3-crystallin (from Tripedalia cystophora) and plant aspartic proteases (from Viridiplantae). Further SAPLIPs used in accordance with the present invention may be bacteriocin AS-48. Bacteriocin AS-48 exhibits antimicrobial activity, can bind to lipids, and has the same fold as the remaining SAPLIP family members but lacks disulfide bridges.

[0086] The present invention is described in further detail below with respect to saposin A as a lipid-binding polypeptide, or a derivative or a truncated form thereof. Saposin A as a lipid-binding polypeptide, or a derivative or a truncated form thereof, is a preferred embodiment, but the present invention should not be limited thereto. Rather, the present invention clearly extends to the entire family of saposin-like proteins (SAPLIPs) as lipid-binding polypeptides of the present invention. Due to the high degree of structural and functional conservation among SAPLIPs, the features and advantages of the specific embodiments of the present invention using saposin A as a lipid-binding polypeptide are expected to apply also to other embodiments using other SAPLIPs or derivatives or truncated forms thereof as lipid-binding polypeptides of the present invention.

[0087] According to a preferred embodiment, the SAPLIP is saposin A, B, C or D, in particular a saposin selected from saposin A, saposin B, saposin C or saposin D of (Homo sapiens, Equus caballus, Bos taurus, Mus musculus, Oryctolagus cuniculus, Rattus norvegicus or Xenopus laevis). In one embodiment, the SAPLIP is saposin A, saposin B or saposin D of (Homo sapiens, Equus caballus, Bos taurus, Mus musculus, Oryctolagus cuniculus, Rattus norvegicus or Xenopus laevis).

[0088] Saposin C is special among saposins in that it can induce membrane fusion, a feature not shown by other saposins. The membrane fusion activity may not always be desirable. According to a specific embodiment of the present invention, the lipid-binding polypeptide is a saposin-like protein (SAPLIP) or a derivative or a truncated form thereof, provided that the SAPLIP is not saposin C, or provided that the SAPLIP is not saposin C or a derivative or a truncated form thereof.

[0089] In one embodiment, the SAPLIP is of human origin (i.e., Homo sapiens SAPLIP).

[0090] In a preferred embodiment, the SAPLIP is saposin A, preferably saposin A of (Homo sapiens, Equus caballus, Bos taurus, Mus musculus, Oryctolagus cuniculus, Rattus norvegicus or Xenopus laevis), particularly preferably human saposin A, the amino acid sequence of which is given as SEQ ID NO: 1. Saposin A is a known protein. Its expression, purification and crystallization as an LDAO-surfactant complex are described, for example, in PNAS, Vol. 109, No. 8 (2012) 2908-2912 (Popovic et al.).

[0091] According to one embodiment of the present invention, the lipid-binding polypeptide comprises the full-length sequence of SAPLIP. In another embodiment, the lipid-binding polypeptide is a derivative of SAPLIP, in particular a polypeptide comprising an amino acid sequence having at least 20, 30, 40, 50 or 60%, preferably at least 75% identity to the full-length sequence of each SAPLIP. In particular, the lipid-binding polypeptide can comprise a sequence having at least 80%, 85%, 90% or 95% identity to the full-length sequence of SAPLIP.

[0092] As used herein, the term "sequence identity" refers to the degree of identity between proteins that can be calculated by optimal alignment of sequences using a scoring matrix such as the Blosum62 matrix described in Henikoff S. and Henikoff JG., P.N.A.S. USA 1992, 89:10915-10919. The calculation of the percentage of identity and optimal alignment of two sequences using the Blosum62 similarity matrix and the Needleman and Wunsch algorithm (J. Mol. Biol. 1970, 48:443-453) can be performed using the GAP program of the Genetics Computer Group (GCG, Madison, WI, USA) with the default parameters of the program.

[0093] As a comparison of amino acid alignments, the EMBL online tool "EMBOSS Stretcher" (http: / / www.ebi.ac.uk / Tools / psa / emboss_stretcher / ) is used with the program in default settings.

[0094] In another embodiment, a derivative of a SAPLIP is a polypeptide comprising a sequence having one or more amino acid deletions, additions, insertions and / or substitutions in the amino acid sequence of each SAPLIP. For example, a SAPLIP derivative may be a polypeptide comprising a sequence of a specific SAPLIP in which 1 to 40, preferably 1 to 30, particularly 1 to 20, or 1 to 15 amino acids are deleted, added, inserted, and / or substituted.

[0095] As used herein, the term "deletion" refers to the removal of 1, 2, 3, 4, 5, or more amino acid residues from each starting sequence.

[0096] As used herein, the terms "insertion" or "addition" refer to the insertion or addition of 1, 2, 3, 4, 5, or more amino acid residues relative to the respective starting sequence. As used herein, the term "substitution" refers to the exchange of an amino acid residue at a particular position with a different position.

[0097] According to another embodiment of the present invention, the lipid-binding polypeptide is a derivative of saposin A comprising one or more fragments of SEQ ID NO: 1. Preferred fragments correspond to the helices a1, a2, a3 and a4 of saposin A, helix a1 being made by the following consecutive stretches of amino acids: "SLPCDICKDVVTAAGDMLK". Helix a2 is made by the following consecutive stretches of amino acids: "ATEEEILVYLEKTCDWL". Helix a3 is made by the following consecutive stretches of amino acids: "PNMSASCKEIVDSYLPVILDIIKGEMS". Helix a4 is made by the following consecutive stretches of amino acids: "PGEVCSAL". According to a particular embodiment of the present invention, the derivative of saposin A is a polypeptide comprising a sequence selected from the helices a1, a2, a3, a4 of saposin A, and combinations thereof, in particular, this polypeptide comprises the sequences of helices a1, a2 and a3 of saposin A. Fragments of saposin A, for example, its helices a1, a2, a3, a4 may have one or more amino acid deletions, additions, insertions and / or substitutions in the amino acid sequence.

[0098] When a derivative of saposin A or a truncated form is used as the lipid-binding polypeptide according to the present invention, when used in the preparation process of the present invention described in detail below, the above-mentioned derivative or truncated form should be amphiphilic, form at least one α-helix, and be able to self-assemble into lipoprotein particles together with solubilized lipids. As used herein, the term "amphiphilic" refers to a polypeptide or molecule that contains both a hydrophilic region and a hydrophobic region.

[0099] Preferably, when a derivative of SAPLIP is used, it has been found that the six cysteine residues corresponding to the six cysteines of SAPLIP should be present in the saposin A members. In this regard, reference is made to the positions of the cysteines in the sequence comparisons of FIGS. 4A and 4B of Bruhn (2005), Biochem J 389(15):249-257, which figure is incorporated herein by reference in particular.

[0100] The lipid-binding polypeptide according to the present invention may also include one or more non-natural amino acids, amino acid analogs, or peptidomimetic structures in which peptide bonds are replaced by structures that are more resistant to metabolic degradation.

[0101] Step a) of the process of the present invention In step a) of the process according to the present invention, unpurified membrane vesicles obtained from the membranes of cells and / or organelles are provided. The unpurified membrane vesicles are always a mixture of different unpurified membrane vesicles. Usually, the unpurified membrane vesicles are not obtained from one cell, but from a plurality of specific types of cells and / or organelles of cells. For example, to obtain unpurified membrane vesicles, cells of a specific eukaryote, prokaryote or archaebacterium may be used. The term "plurality", as used herein, means at least two or more types of cells. It is also possible to use a mixture of cells of different eukaryotes, prokaryotes or archaebacteria to prepare the unpurified membrane vesicles. However, preferably, the unpurified membrane vesicles are obtained from one type of cell or organelle. In another embodiment, the unpurified membrane vesicles are obtained from a single cell. In this embodiment, the process can be carried out at the single-cell level.

[0102] As used herein, the term "unpurified" means that the membrane vesicles are obtained from an unpurified membrane fraction, i.e., they have not been further purified or extracted. Thus, unpurified membranes still contain both membrane proteins and membrane lipids from the membranes of the original cells or organelles from which they are sourced. This is in contrast to prior art processes that primarily use isolated and / or purified proteins or lipids. Unpurified membrane fractions can be obtained by separating insoluble components from soluble components after disruption or lysis of cells or organelles. The insoluble fraction is one form of unpurified membrane fraction that can be used in accordance with the present invention.

[0103] The term "vesicle" is a term of art to those skilled in the art. Typically, a vesicle is a small, circular structure consisting essentially of an aqueous fluid enclosed by a closed, spherical lipid bilayer. However, unpurified membrane vesicles are typically highly diverse in size and content and are heterogeneous. These unpurified membrane vesicles may be specially prepared or may form spontaneously upon disruption or lysis of cells / organelles. The vesicles provided in step a) of the process are obtained from the membranes of cells or organelles. Thus, vesicles typically contain a mixture of the membranes of cells or organelles, particularly membrane lipids and, optionally, membrane proteins from the cells or plurality of cells used.

[0104] The vesicles provided in step a) are a plurality of different vesicles, i.e., a mixture. The vesicles may differ in structure, size, and / or composition. The structure of the vesicles may be monolayer or multilayer. The composition of the vesicles varies depending on the membranes of the cells or organelles from which they are sourced and, if present, the method used in their preparation.

[0105] Certain methods known to those skilled in the art for obtaining vesicles from unpurified membranes may be used (e.g., sonication), and the inventors of the present application have observed that when the membranes of naturally occurring cells or organelles are disrupted or lysed during the disruption or lysis and preparation of unpurified membrane fractions, often a sufficient amount of unpurified membrane vesicles self-assemble.

[0106] The membranes used in the process according to the present invention are selected from cell membranes and organelle membranes. "Membrane", "organelle membrane" or "cell membrane" refers to any membrane containing a lipid layer. Preferably, the membrane is a lipid bilayer. Sometimes, the term "membrane" is used interchangeably herein with "cell membrane" and / or "organelle membrane".

[0107] Basically, the cell membrane is a biological membrane that separates the inside of the cell from the external environment. The complex structure and multiple components (e.g., membrane lipids and membrane proteins) contained in the cell membrane are described in detail in Alberts et al., "The Cell", 4 th edition, Macmillian Magazines Ltd, 2002, pages 583 - 614, and Campbell et al., "Biologie", 6 th edition, Spektrum Verlag, 2003, pages 163 - 177. The cell membrane or organelle membrane used to provide the unpurified membrane vesicles in step a) of the process according to the present invention typically contains a heterogeneous mixture of different lipids and membrane proteins. Thus, the composition of the vesicles provided in step a) may differ in terms of being a mixture of membrane lipids and membrane proteins and typically varies depending on the particular membrane that is the source.

[0108] The terms "lipid" or "membrane lipid", as used herein, are recognized in the art and refer to naturally occurring substances derived from living organisms that are soluble or partially soluble in organic solvents or that partition into hydrophobic environments when present in an aqueous phase. Sometimes the terms "lipid" and "membrane lipid" are used interchangeably herein. The terms "lipid" or "membrane lipid", as used herein, do not mean a synthetic molecule or a single type of lipid molecule in the particles of the present invention. In fact, this term means a plurality of at least two different lipid molecules present in the membranes of cells or organelles. The lipids incorporated into the sariplo particles of the present invention are membrane lipids that naturally occur in the membranes of cells or organelles. Thus, the particles of the present invention typically contain a mixture of membrane lipids that naturally occur in the membranes of the source cells or organelles. In one embodiment, the particles obtained according to the present invention contain at least 3, 5, 10 or 20 different lipids. Typically, these membrane lipids form a bilayer in which membrane proteins are embedded. Exceptions are given for some archaeal membranes, since some archaea contain a monolayer and can thus be used in the processes according to the present invention. The structure of the archaeal cell membrane is described in more detail below.

[0109] Cell membranes or organelle membranes basically contain three types of amphiphilic lipids as membrane lipids: phospholipids, glycolipids, or sterols. The amount of each varies depending on the type of cell, cell membrane, or organelle membrane. Phospholipids have a polar part (phosphate "head") that dissolves in water and a hydrophobic non-polar part ("lipid tail") that does not dissolve in water. These parts are connected by a glycerol moiety. In water, phospholipids can form clusters where the heads face the water and the tails face away from the water. The fatty acid chains of phospholipids and glycolipids usually contain an even number of carbon atoms, typically 16 - 20 carbon atoms. Fatty acids with 16 and 18 carbon atoms are the most common. Fatty acids can be either saturated or unsaturated. The conformation of the double bond is typically in the so-called cis conformation. Cis- and trans- isomers are terms used in organic chemistry to refer to stereoisomers that occur in the relative arrangement of functional groups within a molecule according to Cahn-Ingold-Prelog (CIP; Cahn, R.S. & Ingold, C.K.; Prelog, V., "Specification of Molecular Chirality". Angewandte Chemie International Edition, 5(4), p.385 - 415, 1966). Typical fatty acids present in cell membranes are also described on pages 61 and 62 of Alberts et al., "The Cell", 4 th edition, Macmillian Magazines Ltd, 2002. Further examples of membrane lipids are phospholipids such as phosphatidylcholine such as POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), phosphatidylethanolamine, and phosphatidylserine such as POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine) phosphatidylinositol, and sphingomyelin.

[0110] Glycolipids are lipids to which carbohydrates are attached by glycosidic bonds. Carbohydrates are typically found on the outer surface of the cell membranes of eukaryotes. These carbohydrates extend from the phospholipid bilayer into the aqueous environment outside the cell. Examples of glycolipids are glyceroglycolipids, galactolipids, sulfolipids, glycosphingolipids, glucocerebrosides, sulfatides, gangliosides, globosides, glycophosphosphingolipids, and glycosylphosphatidylinositol. Sterols are a subgroup of steroids. Sterols as part of membranes typically occur within the membranes of eukaryotes such as plants, animals, and fungi. Examples of sterols are cholesterol, campesterol, sitosterol, stigmasterol, and ergosterol. According to a preferred embodiment, the membrane lipid is a lipid that forms a lipid bilayer and / or a biocompatible lipid. The term "biocompatible" as used herein indicates being biologically compatible by not causing toxicity, harmfulness, or an immunological response in living cells.

[0111] As used herein, "lipid that forms a bilayer" refers to a lipid that can form a lipid bilayer with a hydrophobic inner side and a hydrophilic outer side. Any lipid that can associate with SAPLIP, or its derivatives or truncated forms, and aggregate into a particle structure may be used according to the present invention. Lipids that form a bilayer include, but are not limited to, phospholipids, sphingolipids, glycolipids, alkylphospholipids, ether lipids, and plasmalogens. One type of lipid that forms a bilayer may be used, or a mixture of two or more types may be used. The particles may contain lipids that are not lipids forming the bilayer. Such lipids include, but are not limited to, cholesterol, cardiolipin, phosphatidylethanolamine (which can form a bilayer under certain circumstances), oxysterol, phytosterol, ergosterol, sitosterol, cationic lipids, cerebrosides, sphingosine, ceramide, diacylglycerol, monoacylglycerol, triacylglycerol, ganglioside, ether lipids, alkylphospholipids, plasmalogens, prostaglandins, and lysophospholipids.

[0112] The membrane lipids contained in the sariplo particles of the present invention may be a mixture of the membrane lipids listed above, but are not limited thereto. Typically, the membrane lipids contained in the particles according to the present invention include at least phospholipids, glycolipids, cholesterol, and mixtures thereof. According to a preferred embodiment, the membrane lipids are eukaryotic lipids and / or prokaryotic lipids, and in particular, typically, those present in any one of the membranes present in eukaryotic or prokaryotic cells. Preferred lipids are, for example, phospholipids, glycosphingolipids, sterols, phosphatidylcholine, phosphatidylserine (PS), 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC), 2-oleoyl-1-palmitoyl-sn-glycero-3-glycerol (POPG), 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphoethanolamine (POPE), diacylglycerol, cholesterol, sphingomyelin, galactosylceramide, ganglioside, phosphatidylinositol, and sulfoglycosphingolipid, or combinations thereof.

[0113] In another embodiment, the membrane lipid comprises a phospholipid. Examples of suitable phospholipids include, but are not limited to, DMPC, DMPG, POPC, dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylserine (DPPS), cardiolipin, dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), egg yolk phosphatidylcholine (egg PC), soy phosphatidylcholine, phosphatidylinositol, phosphatidic acid, sphingomyelin, and cationic lipids. The membrane lipids in the particles according to the present invention are typically a heterogeneous mixture of lipids present in the membranes of cells or organelles. However, it is also possible for the particles of the present invention to further contain lipids, which may be modified lipids containing one or more attached functional moieties, such as targeting moieties or bioactive moieties.

[0114] The targeting moiety may, for example, be useful when the particles of the present invention target a specific cell or tissue type, or an infectious agent. The targeting moiety may also be useful for purifying, testing, or identifying the particles. In some embodiments, the particles comprise a targeting moiety attached to a lipid-binding polypeptide or a lipid component or a membrane protein component.

[0115] The targeting moiety may, for example, have receptor recognition properties so that the particles can target a specific cell surface receptor. For example, the particles of the present invention may have a specific type of infectious agent by modifying, for example, the lipid-binding polypeptide component of the particles so that it can interact with a receptor on the surface of the targeted cell type. In one embodiment, the targeting moiety is selected from the group consisting of natural or synthetic ligands, antibodies and antibody fragments, or other biomolecules suitable for targeting purposes.

[0116] The bioactive moiety may be selected, for example, from drugs, cytotoxic agents, enzymes, labels, fluorophores, contrast agents, and radioisotopes.

[0117] In addition to the membrane lipids of the membranes of cells or organelles, the salipro particles of the present invention may further contain additional lipids. These lipids may be selected from naturally occurring lipids, synthetic lipids, modified lipids, fats, waxes, sterols, fat-soluble vitamins, monoglycerides, diglycerides, triglycerides, phospholipids, fatty acids, glycerolipids, glycerophospholipids, sphingolipids, glycolipids, polyketides, sterol lipids and prenol lipids, or combinations thereof.

[0118] The salipro particles according to the present invention may lack membrane proteins, i.e., may consist essentially of only lipid-bound polypeptides and membrane lipids from unpurified membrane vesicles ("empty" salipro particles). However, preferably, the salipro particles also contain membrane proteins ("filled" salipro particles). Membrane proteins are proteins that are naturally embedded in the membrane or, optimally, proteins that associate only with the membrane. Membrane proteins can exhibit various functions. For example, membrane receptor proteins coordinate signals between the inner and outer environments of the cell, and transport proteins move molecules and ions across the membrane. Membrane proteins may act as enzymes, which may have many activities (e.g., oxidoreductase, transferase or hydrolase activities). Membrane proteins may be, for example, cell adhesion molecules.

[0119] According to one embodiment, the salipro particles of the present invention do not contain membrane proteins or contain 1 to 10, or 1 to 5, membrane proteins per particle. A library can also be defined, according to one embodiment, as containing particles with an average of 1 to 10 or 1 to 5 membrane proteins per particle.

[0120] The membrane proteins in the particles of the present invention may be selected from, for example, integral membrane proteins, integral transmembrane proteins, integral single-pass transmembrane proteins, peripheral membrane proteins, amphitropic proteins in a lipid-bound state, proteins immobilized on lipids, and chimeric proteins having fused hydrophobic and / or transmembrane domains.

[0121] Integral membrane proteins are membrane proteins that are permanently bound to the lipid bilayer and usually require a surfactant or nonpolar solvent to move and form a membrane. Transmembrane proteins are integral membrane proteins that extend across the membrane at least once. Examples of transmembrane proteins that can be incorporated into the particles of the present invention are G protein-coupled receptors (GPCRs), transporters, such as uniporters, symporters or antiporters, channels, such as ion channels or enzymes.

[0122] Integral single-pass transmembrane proteins are permanently connected to the membrane only from one side and do not extend across the entire membrane. This type includes membrane proteins that are fixed to the membrane via an α-helical transmembrane anchor. Examples include cytochrome P450 oxidase and glycophorin A.

[0123] Peripheral membrane proteins associate with the lipid bilayer or integral membrane proteins incorporated therein only temporarily or indirectly. Peripheral membrane proteins usually dissociate from the membrane after treatment with a polar reagent at high pH or high salt concentration. Examples of peripheral membrane proteins include phospholipase A2 or C, lipoxygenase and cytochrome c.

[0124] Proteins immobilized on lipids bind to the lipid bilayer by lipid-imparted, particularly prenylated or GPI-immobilized amino acid residues. Examples include bacterial lipoproteins, G proteins and certain kinases.

[0125] An amphitropic protein is a protein that exists in at least two conformational states: a lipid-free aqueous state and a lipid-bound state. When it associates with lipids, the amphitropic protein undergoes a conformational change and becomes associated with the membrane either reversibly or irreversibly. Examples of amphitropic proteins are pore-forming toxins and antibacterial peptides.

[0126] The vesicles provided in step a) may be obtained from cells and / or cell organelles of the cell. The term "one (a)" as used herein means "one" or "a plurality of" specific objects. For example, "a cell" means one specific cell or a plurality of specific cells. Typically, a plurality of cells and / or cell organelles of the cell are used in the process according to the present invention. The process according to the present invention is not limited to the use of one cell and / or cell organelles of the cell. It is also possible to use a mixture of different cells or cell organelles to prepare membrane vesicles.

[0127] In a particular embodiment of the present invention, the unpurified membrane vesicles of step a) are a.1) a step of providing cells and / or cell organelles of the cell; a.2) a step of lysing or disrupting the cells and / or the cell organelles of the cell; a.3) a step of obtaining an unpurified membrane fraction; and a.4) a step of preparing unpurified membrane vesicles from the unpurified membrane fraction obtained in step a.3), and are prepared by at least one, two, three, or all of them.

[0128] In step a.1), cells and / or cell organelles of the cell are provided. The term "cell" as used herein is understood to be typically the same as that used in biology. A cell is the basic structural unit, functional unit, and biological unit of all known living organisms. A cell is the smallest unit of life that can replicate independently. "Cell" as used herein includes eukaryotes, prokaryotes, and archaea. "Cell" as used herein does not include viruses. Cells contain cytoplasm enclosed by a cell membrane, which, as described above, contains biomolecules such as proteins and lipids. Organisms may be classified as unicellular (i.e., consisting of a single cell, e.g., prokaryotes) or multicellular (e.g., eukaryotes, such as animals, plants, or fungi).

[0129] The cells from which the membranes used in the process according to the present invention are obtained are not limited to specific types of cells. The cells may be naturally occurring, transfected, genetically engineered, or diseased cells, such as cancer cells. It has been found that various types of cells are suitable for the process according to the present invention. Preferably, the cells are cells of archaea, eukaryotes, or prokaryotes. Preferred cell organelles are those of eukaryotic cells.

[0130] The cell membranes of archaea, eukaryotes, and prokaryotes each contain the membrane lipids described above and optionally membrane proteins. However, those skilled in the art know that the cells of archaea, eukaryotes, and prokaryotes are different from each other. The structures, functions, and differences of the cells of archaea, eukaryotes, or prokaryotes are described in various standard textbooks, e.g., Alberts et al., "The Cell", 4 th edition, Macmillian Magazines Ltd, 2002 or Campbell et al., "Biologie", 6 th edition, Spektrum Verlag, 2003.

[0131] Eukaryotes are any cells or organisms in which the cells contain a nucleus and optionally membrane-enclosed organelles. In one embodiment, the membranes used in the process according to the present invention are membranes from eukaryotes, e.g., cell membranes and / or membranes derived from the cell organelles of cells. Examples of cell organelles are the Golgi apparatus, mitochondria, peroxisomes, endoplasmic reticulum, chloroplasts, nuclei, etc.

[0132] Examples of eukaryotes are plants, animals and fungi, such as yeast and mold. Preferred eukaryotic cells that can be used in the process according to the present invention are selected from the group consisting of mammalian cells, particularly animal and human cells, insect cells, avian cells, fungal cells, such as yeast cells, plant cells, and mixtures thereof. The term mammalian cells particularly also includes animal and human cells maintained in culture medium.

[0133] Prokaryotes are single-celled organisms without a nucleus. Prokaryotic cells are simpler and smaller than eukaryotic cells and do not have membrane-bound organelles. Examples of prokaryotes are bacteria. Exemplary bacterial phyla are Acidobacterium, Actinomycetes, Aquifex, Almatimonadaceae, Bacteroidetes, Caldisericum, Chlamydia, Chlorobi, Chloroflexi, Chrysiogenes, Cyanobacteria, Deferribacter, Deinococcus-Thermus, Dictyoglomus, Elusimicrobia, Fibrobacter, Firmicutes, Fusobacterium, Gemmatimonadetes, Lentisphaerae, Nitrospira, Planctomycetes, Proteobacteria, Spirochaetes, Synergistetes, Tenericutes, Thermodesulfobacteria, Thermotoga and Verrucomicrobia. Preferred prokaryotic cells that can be used in the process according to the present invention are bacteria, particularly pathogenic bacteria, and mixtures thereof.

[0134] Archaea are only remotely related to prokaryotes and eukaryotes. A detailed overview is given, for example, in De Rosa et al., "Structure, Biosynthesis, and Physicochemical Properties of Archaebacterial Lipids", MICROBIOLOGICAL REVIEWS, p. 70-80 Vol. 50, No. 1, 1986 or Albers et al., "The archaeal cell envelope", Nature Reviews Microbiology, 9, p. 414-426, 2011.

[0135] De Rosa et al. have reported that archaeal membranes contain molecules that are very different from those of prokaryotes and eukaryotes. Prokaryotes and eukaryotes contain membranes that mainly consist of glycerol ester lipids, while archaea contain membranes that consist of glycerol ether lipids. Ether bonds are chemically more resistant than ester bonds. This stability would help archaea survive in extreme temperatures and very acidic or basic environments. Although prokaryotes and eukaryotes may contain ether lipids, in contrast to archaea, these lipids are either a minor component of the membrane or not included as a membrane component.

[0136] In addition to this, archaeal lipids are based on isoprenoid side chains. Isoprenoid side chains are long chains containing up to 20, 25 or 40 carbon atoms and may optionally have branches of multiple side chains. Isoprenoid side chains may also contain a cyclopropane ring or a cyclohexane ring. This is in contrast to the fatty acids found in the membranes of the other organisms described above. Isoprenoids play an important role in the biochemistry of many organisms, but only archaea use this for the production of phospholipids. In certain archaea, the lipid bilayer may be replaced by a monolayer.

[0137] Examples of archaea are methanogenic archaea, halobacteria and thermoacidophilic archaea. Preferred archaea that can be used in the process according to the invention are restricted archaea or mixtures of different restricted archaea.

[0138] Using viruses that may contain lipids or proteins from the host is not part of the present invention, which is directed only to vesicles and particles derived from the membranes of cells or organelles. The terms cell or organelle exclude viruses. The viral structure and the components of the viral membrane are, where applicable, different from the membranes of eukaryotes, prokaryotes and archaea. This is reported in more detail, for example, in Lorizate et al., "Comparative lipidomics analysis of HIV-1 particles and their producer cell membrane in different cell lines", Cellular Microbiology, 15(2), p.292-304, 2013 and Bruegger et al., "The HIV lipidome: A raft with an unusual composition", PNAS, Vol.103, No.8, p.2641-2646, 2006.

[0139] Lorizate et al. reported that various tests showed that the HIV-1 membrane is different from the producer cell plasma membrane, suggesting viral budding from existing subdomains or virus-mediated induction of a special budding membrane. From the lipid analysis of plasma membranes and HIV-1 purified from two different cell lines, it was found that this viral membrane has a significantly different lipid composition independent of the cell type observed, compared to the plasma membrane of the host cell. Viral particles were significantly enriched in phosphatidylserine, sphingomyelin, hexosylceramide, and saturated phosphatidylcholine species compared to the host plasma membrane of the producer cells. These showed a decrease in the levels of phosphatidylethanolamine and phosphatidylinositol, which are unsaturated phosphatidylcholine species. Cell type-specific differences in the lipid composition between HIV-1 and the donor plasma membrane were observed for plasmalogen-phosphatidylethanolamine and phosphatidylglycerol, which were very abundant only in HIV-1 derived from MT-4 cells. HIV-1 derived from MT-4 cells also contained dihydrosphingomyelin. Together, these data reported by Lorizate et al. support that HIV-1 selects a special lipid environment for its morphogenesis and is not identical or even similar to the host cell membrane. Usually, the particles and libraries of the present invention described herein do not contain viral proteins and / or viral membranes.

[0140] The cells of step a.1) may be provided, for example, as a purified cell fraction or an unpurified cell fraction, such as a cell suspension or a growth medium. The cells may be provided in the form of a cell culture, and the cells typically grow in a controlled state (e.g., inside or outside their natural environment). Those skilled in the art know that the cells in a cell line are often very similar but not identical. However, providing a plurality of cells obtained from a certain cell line is also included in the meaning of providing cells in step a.1). The cells or organelles cited in step a.1) may be part of a larger and more complex group of the cells and / or organelles used in this step.

[0141] In step a.1), the cells may be provided in the form of an unpurified biological sample, such as a sample containing tissue, an organ, or any other biological cell. However, those skilled in the art may isolate the cells, the cell organelles of the cells, or a plurality of these from a sample (e.g., a cell culture, tissue, tissue suspension, or any other biological sample).

[0142] There are several techniques available for separating different cell types from a mixed cell suspension. One is to utilize the differences in physical properties. By centrifugation, large cells may be separated from, for example, small cells, and high-density cells may be separated from light cells.

[0143] Another approach is based on the tendency of some cell types to adhere strongly to glass or plastic, allowing these cells to be separated from those that do not adhere as strongly. Additional cell separation techniques use antibodies conjugated to dyes, labels, or tags that target specific cells. The labeled cells can then be separated from the unlabeled cells, for example, by FACS or other methods. Specific cells can also be obtained by carefully excising them from tissue sections. Selected cells can be isolated from tissue sections by microdissection techniques. This method may include, for example, a laser beam that excites the area of interest and releases it into a container, allowing isolation of even a single cell from a tissue sample.

[0144] Various techniques for the separation, purification, or isolation of cells and cell organelles may be used in the methods of the present invention. The above-described methods in terms of cells are also suitable for obtaining cell organelles of the cells.

[0145] In step a.2), at least a part of the cells and / or cell organelles of the cells are disrupted or lysed. The terms "lyse" or "disrupt" refer to breaking at least a part of the membrane of the cell or cell organelle. The cells and / or cell organelles of the cells can be lysed / disrupted by chemical means or mechanical means. Depending on the lysis / disruption technique used, all, some, or only a part of the membrane may be lysed / disrupted. For example, when only the cell membrane is lysed / disrupted, gradient centrifugation may be used to collect specific cell organelles.

[0146] Lysis or disruption may be carried out enzymatically, by chemical agents, or mechanically. Repeated freeze-thaw cycles, sonication, e.g., ultrasonication, pressure, disruption by hypoosmotic shock, or mechanical lysis of cell or organelle membranes by filtration are sometimes also called lysis. The untreated solution immediately after lysis, but before any further extraction or purification steps, is called the crude lysate. From the crude lysate, a crude membrane fraction containing the membranes present in the lysate can be isolated. Typically, this can be achieved by separating the insoluble fraction (crude membrane fraction) of the lysate from the soluble fraction, e.g., by centrifugation. In a preferred embodiment of the invention, the crude membrane vesicles are obtained from the crude membrane fraction without performing any other vesicle generation, purification or extraction steps.

[0147] In step a.3), a fraction of the crude membrane is obtained. In one embodiment of the invention, steps a.2) and a.3) may be carried out simultaneously. This can be achieved, for example, by lysing the cells and / or cell organelles of the cells, thereby giving crude membrane vesicles.

[0148] In step a.4), the crude membrane vesicles can be obtained from the crude membrane fraction after step a.3), or after steps a.2) and a.3). The crude membrane vesicles can be obtained by partially dissolving the crude membrane fraction in a solvent, buffer, surfactant or a mixture thereof, or by agitation. Examples of suitable aqueous solvents are water, e.g., deionized water, desalted water or sterile water, physiological saline solutions, e.g., NaCl solution, and solutions containing phosphates, acetates, glycine salts, ammonium salts, calcium salts, magnesium salts, potassium salts, or mixtures thereof. Preferred buffers are physiologically tolerable buffers. Examples of suitable buffers are, for example, phosphate buffer, ACES, PIPES, imidazole / HCl, BES, MOPS, HEPES, TES, TRIS, HEPPS or TRICIN.

[0149] In another embodiment of the present invention, steps a.2), a.3) and / or a.4) are carried out simultaneously. By lysing the cells and / or cell organelles of the cells, the crude membrane fraction is present in the lysate. The inventors of the present application have observed that in many cases, crude membrane vesicles are spontaneously formed and used in the process of the present invention.

[0150] Step b) of the process of the present invention In step b) of the method of the present invention, the mixture obtained after step a) (i.e., the crude membrane vesicles) is contacted with a lipid-binding polypeptide in a liquid environment. Step b) of the method of the present invention may be formulated as "contacting the crude membrane vesicles with a lipid-binding polypeptide in a liquid environment". The crude membrane vesicles are those obtained in step a) and / or a.4).

[0151] The lipid-binding polypeptide and the lipid may be in the forms described above.

[0152] In certain embodiments, the liquid environment is an aqueous solution. The aqueous solvent may be a buffered solution. The liquid environment of step b) may have a pH of 2.0 to 10.0, particularly 6.0 to 10.0, preferably 6.0 to 9.0, particularly preferably 7.0 to 9.0, and most preferably 7.0 to 8.0. The liquid environment of step b) may optionally also contain a surfactant.

[0153] The duration of steps b) and / or c) depends very much on the specific experimental setup, particularly the types of lipids and membrane proteins present in the sariplo particles and the temperature at which these steps are carried out. Good results have generally been achieved when steps b) and c) each continue independently of each other for 10 seconds to 24 hours, 10 seconds to 2 hours, 10 seconds to 30 minutes, 10 seconds to 15 minutes. Preferably, steps b) and / or c) are carried out at a temperature of 10 to 60 °C, preferably 15 to 42 °C, particularly preferably 30 to 40 °C. However, it is also possible to work at a lower temperature (e.g., 2 to 10 °C) when the incubation time is increased. The temperatures shown are the temperatures of the liquid environment in which the steps are carried out.

[0154] In certain embodiments of the process according to the invention, the crude membrane vesicles are contacted with a surfactant in step a), b) and / or c).

[0155] The term "surfactant", as used herein, is recognized in the art and is not included in the definition of "lipid" or "membrane lipid" as used herein.

[0156] Many lipids have a similar general amphiphilic structure compared to surfactants, i.e., a polar hydrophilic head group and a nonpolar hydrophobic tail, but lipids differ from surfactants in terms of the shape of the monomer, the type of aggregates formed in solution, and the concentration range required for aggregation. Lipids generally have a substantially cylindrical structure, and the volume occupied by the hydrophobic tail is similar to the volume occupied by the polar head group. Surfactant monomers are generally more conical in shape, and the volume occupied by the hydrophobic tail is smaller than the volume occupied by the polar head group. Surfactants tend to aggregate into spherical or ellipsoidal micelles, which are water-soluble and do not form bilayer structures in the absence of lipids (see handbook "Detergents and their uses in membrane protein science", Anatrace, www.anatrace.com). Surfactants that can be used in the process according to the invention may be anionic, cationic, nonionic, zwitterionic and mixtures thereof.

[0157] The surfactants used in this specification are preferably selected from the group consisting of alkylbenzene sulfonates or bile acids, cationic surfactants, and nonionic or zwitterionic surfactants, such as lauryl-dimethylamine-oxide (LDAO), Fos-choline, CHAPS / CHAPSO, alkyl glycosides, such as short-chain, medium-chain or long-chain alkyl maltosides, especially n-dodecyl β-D-maltoside, glucoside, maltose-neopentyl glycol (MNG) amphiphilic molecules, amphiphilic polymers (amphipols), macrocyclic or cyclic oligomers (calixarenes) based on hydroxyalkylated products of phenols and aldehydes, and mixtures thereof.

[0158] Typical anionic surfactants are alkylbenzene sulfonates. The alkylbenzene moiety of these anions is lipophilic and the sulfonate is hydrophilic. The anionic surfactant may contain, for example, a branched alkyl group or a straight-chain alkyl group. Examples of suitable anionic surfactants are bile acids, such as deoxycholic acid (DOC), alkylbenzene sulfonates, such as sodium branched dodecylbenzenesulfonate, sodium linear dodecylbenzenesulfonate, and mixtures thereof.

[0159] Cationic surfactants are very similar to anionic surfactants and have a hydrophobic component, but instead of an anionic sulfonate group, the cationic surfactant has a quaternary ammonium at the polar end. The center of the ammonium is positively charged.

[0160] Non-ionic surfactants are characterized by uncharged hydrophilic head groups. Typical non-ionic surfactants are based on, for example, polyoxyethylene or glycosides. Common examples of the former include the Tween, Triton, and Brij series. These materials are also known as ethoxylates or PEG-reates and the metabolite nonylphenol. Glycosides have a sugar as an uncharged hydrophilic head group. Examples include octylthioglucoside and maltoside. The surfactants of the HEGA series and MEGA series are closely related and have a sugar alcohol as the head group. Examples of suitable non-ionic surfactants that can be used in the process according to the present invention are alkyl glycosides, such as short-chain, medium-chain, or long-chain alkyl maltosides, such as n-dodecyl-β-maltoside (DDM), decanoyl-N-hydroxyethyl glucamide (HEGA), n-decanoyl-N-methyl-D-glucamide (MEGA), and mixtures thereof.

[0161] Amphoteric surfactants have a net zero charge resulting from the presence of an equal number of oppositely charged chemical groups, i.e., the total number of negative charges and the total number of positive charges are equal so that the overall charge is net zero. Examples include Fos-choline, CHAPS / CHAPSO, lauryl-dimethylamine-oxide (LDAO), and mixtures thereof.

[0162] From actual experiments, it has been shown that it is advantageous to use surfactants having short-chain to medium-chain hydrophobic tails. This is particularly true when membrane proteins are incorporated as hydrophobic agents. "Short-chain hydrophobic tail" as used herein means, for example, C2 to C9 in n-nonyl-β-maltoside (NM). "Medium-chain hydrophobic tail" as used herein means, for example, C10 to C15 in n-decyl-β-maltoside (DM) or n-dodecyl-β-maltoside (DDM). In one embodiment, the surfactant used for the purification and / or solubilization of the hydrophobic agent contains 2 to 12 carbon atoms in its hydrophobic tail, preferably 2 to 10, most preferably 2 to 9 carbon atoms in its hydrophobic tail.

[0163] The membrane vesicles in any one of step a) or a.1), a.2), a.3) and / or a.4) may be in a solubilized state with a surfactant. Actual experiments have shown that a wide variety of surfactants can be used and the unpurified membrane vesicles can be further solubilized for use in the process according to the present invention. For example, the process according to the present invention functions very well using unpurified membrane vesicles present in a solution containing alkyl glucosides such as 0.01 to 5.0%, especially 0.1 to 1.0% short-chain or longer-chain alkyl maltosides and glucosides. However, depending on the type of membrane or unpurified membrane vesicles used, other suitable surfactants can equally be used. The ability of a given surfactant to solubilize a given membrane or unpurified membrane vesicles can be easily visually observed by the formation of a clear solution without aggregates, precipitation or phase separation. Without being bound by theory, the addition of a surfactant seems to loosen the membrane structure and assist in the self-assembly of the particles, i.e., the unpurified membrane components seem to be incorporated into the sarp particles.

[0164] In one embodiment of the present invention, the surfactant used to dissolve the mixture in step b) is not retained in a significant amount in the final particles of the present invention. In particular, the amount of surfactant in the particles obtainable by the process according to the present invention can be made undetectably low. In one embodiment, the particles of the present invention do not contain a significant amount of surfactant, and in particular, contain less than 0.1 wt%, preferably less than 0.01 wt%, particularly preferably less than 0.001 wt% of surfactant, based on the weight of the particles. The amount of surfactant present in the particles can be determined, for example, by mass spectrometry.

[0165] Actual experiments have shown that the lipid-conjugated polypeptides of the present invention generally do not require surfactants or other solvents during purification, storage or handling. Optionally, however, the lipid-conjugated polypeptides used in step b) may also be in a solubilized form with a surfactant.

[0166] In some embodiments, the molar ratio of the lipid-conjugated polypeptide to the membrane vesicles in step b) is at least 1:1, preferably at least 2:1 or at least 5:1, particularly preferably at least 10:1. In another embodiment, the molar ratio of the lipid-conjugated polypeptide to the membrane vesicles in step b) is from 1:1 to 1,000,000:1, particularly from 1:1 to 100,000:1, or from 10,000:1 to 500:1.

[0167] In some embodiments, the weight ratio of the lipid-conjugated polypeptide to the membrane vesicles in step b) is at least 1:1, preferably at least 2:1 or at least 5:1, particularly preferably at least 10:1. In another embodiment, the weight ratio of the lipid-conjugated polypeptide to the membrane vesicles in step b) is from 1:1 to 1,000,000:1, particularly from 1:1 to 100,000:1, or from 10,000:1 to 500:1.

[0168] In one embodiment, the lipid-binding polypeptide in step b) is in molar excess compared to the unpurified membrane vesicles. In another embodiment, the amount of the lipid-binding polypeptide in step b) is in excess compared to the unpurified membrane vesicles (the wt% at each time is based on the total weight of the mixture in step b).

[0169] For most of the membrane proteins incorporated into the particles of the present invention, optimal results are achieved when the molar ratio of lipid to membrane protein in step b) is from 100,000:1 to 1000:1, particularly from 100:1 to 1:1.

[0170] In one embodiment of the process according to the present invention, the molar ratio of the lipid-binding polypeptide to the membrane vesicles in step b) is at least 1:1, particularly at least 3:1, preferably at least 5:1 or 10:1. In another embodiment of the process according to the present invention, the weight ratio of the lipid-binding polypeptide to the membrane vesicles in step b) is at least 1:1, particularly at least 3:1, preferably at least 5:1 or 10:1.

[0171] In another embodiment, steps a) and b) are carried out simultaneously. In a further embodiment, at least one of steps a.1) to a.4) is carried out simultaneously with step b). In this embodiment, it is also possible to carry out at least two of steps a.1) to a.4) and step b) simultaneously. In a particular embodiment, all of steps a.1) to a.4) and step b) are carried out simultaneously. In a preferred embodiment, steps a.3), a.4) and step b) are carried out simultaneously.

[0172] In a further embodiment of the present invention, the process further comprises, between step a) and step b), b.1) contacting the unpurified membrane vesicles with a surfactant in a liquid environment. The surfactant may be as defined above.

[0173] When step b.1) is carried out, the mixture obtained after step b.1) (i.e., the mixture of unpurified membrane vesicles and surfactant) is contacted with the lipid-bound polypeptide in step c). Optionally, between step b.1) and step b), step b.2) may also be present. After purifying the mixture of unpurified membrane vesicles and surfactant obtained in step b.1), it is contacted with the lipid-bound polypeptide in step b). Such purification is described in more detail below and may include, for example, removal of the surfactant, especially removal of excess or substantially all of the surfactant, or removal of non-vesicle components. An example of such purification is the removal of test pieces and / or protein aggregates by ultracentrifugation. When step b.2) is carried out, the mixture obtained after step b.2) (i.e., the purified mixture of unpurified membrane vesicles) is contacted with the lipid-bound polypeptide in step c).

[0174] Such a purification step may be carried out as step c.1) between steps b) and c), and the mixture obtained after steps a) and b) is purified.

[0175] Steps a), b) and / or c) may be carried out in the presence of a surfactant. Preferably, the surfactant is selected from the surfactants described above. The surfactant may be added in a suitable form such as a liquid, solid or from a liquid to a solid state.

[0176] In certain embodiments, the mixture obtained after step a), a.4), b) or b.1) is optionally purified. Such purification steps are described, for example, as steps b.2) and c.1) above.

[0177] Suitable purification methods include chromatography methods, especially size exclusion chromatography, ultracentrifugation, dialysis, contact with biobeads that bind to the surfactant, use of concentrators, affinity chromatography for removing unbound / incorporated lipids and / or hydrophobic compounds, magnetic beads and / or membranes / filters. One of ordinary skill in the art can select a suitable purification method depending on the purification goal to be achieved.

[0178] Step c) of the process of the present invention In step c) of the process according to the present invention, self-assembly of the particles occurs. Step c) does not need to be a separate step and can be carried out simultaneously with step b). Usually, self-assembly of the particles occurs immediately upon contacting the crude membrane with the lipid-binding polypeptide of the present invention. Step c) is usually carried out in a liquid environment, which may be the same as or different from the liquid environment used in step b).

[0179] Self-assembly of the particles in step b) may be referred to as fragmentation of the crude membrane or crude membrane vesicles into the sariplo particles of the present invention. This fragmentation or self-assembly occurs by contacting with the lipid-binding polypeptide of the present invention.

[0180] Preferably, step c) of the present invention comprises self-assembling the particles at a pH of 2.0 to 10.0, particularly 6.0 to 10.0, preferably 6.0 to 9.0, particularly preferably 7.0 to 9.0, most preferably 7.0 to 8.0. These pH ranges may be applied to step b) described above, independently of the pH applied to step c). By the pH ranges described herein, the components can be brought into contact with each other in step b) in order to self-assemble particularly effectively into the particles of the present invention in step c).

[0181] Contrary to the expectation that the sarpuro particles should aggregate best at a pH of 4.75, which is the optimal value of the natural pH of saposin, or at a pH close thereto, it has been found that a library of sarpuro particles exhibiting improved properties and an extended spectrum of uses can also be obtained if a more neutral or basic pH is maintained during the self-assembly of the particles. Surprisingly, at a pH of 5.0 to 10 (especially 6.0 to 10, more preferably 6.0 to 8.5, and most preferably 7.0 to 8.0), in the presence of unpurified membrane vesicles, a purified saposin-like protein or its derivative or a truncated form thereof can self-assemble into stable lipoprotein particles without requiring a cumbersome upstream purification process for either the lipid or membrane protein components to be incorporated.

[0182] Step c) of the process according to the invention may comprise, or may consist of, diluting the mixture obtained in step b) with a liquid, in particular a liquid that does not contain a surfactant or contains a smaller amount of surfactant than the mixture obtained in step b). Actual experiments have shown that such a dilution step further induces and promotes the self-assembly of the particles of the invention. Without wishing to be bound by this theory, it is believed that such a dilution step effectively removes impurities, solvents and / or surfactant molecules from the hydrophobic surfaces of the lipid-bound polypeptide and the unpurified membrane vesicles, thereby facilitating the particle self-assembly process according to the invention by enhanced hydrophobic interactions of the components.

[0183] The self-assembly of the particles in step c) may be carried out with exactly the same composition as that prepared in step b), but step c) may also include, or may consist of, the addition of an organic solvent, the removal of a surfactant, a purification or dilution step. Step c) may be, for example, a gel filtration step. In this case, the mixture obtained after step b) is purified and diluted in step c) with the gel filtration buffer used. In certain embodiments, a gel filtration step is performed on the mixture obtained in step b), whereby the gel filtration buffer or other solution is a liquid that does not contain a surfactant or contains a lower amount of surfactant than the mixture obtained in step b).

[0184] According to certain embodiments, one of the above-described steps, in particular steps a), b) and / or c) and / or the respective sub-steps a.1) to a.4, b.1) to b.2) and / or c.1) described therein, is carried out at a temperature of 4 °C to 85 °C, in particular 20 °C to 70 °C, particularly preferably 30 °C to 70 °C. For most applications, a temperature of 30 °C to 40 °C is sufficient. However, by the methods taught herein, one skilled in the art can determine the optimal incubation temperature with respect to the temperature stability of the membranes, compounds, lipids and proteins used.

[0185] In a further embodiment, the process includes purifying the particles in step c) or as a subsequent step d) by at least partially removing free membrane lipids, free membrane proteins, free lipid-binding polypeptides, insoluble or aggregated substances and / or surfactants, optionally the purification being carried out by chromatography, in particular size exclusion chromatography; ultracentrifugation; dialysis; contact with biobeads that bind surfactants; use of a concentrator; chromatography, magnetic beads, immunoaffinity purification and / or affinity purification methods including membranes / filters for removing unbound / incorporated lipids and / or hydrophobic compounds, without limitation.

[0186] By the above process, a library of sarp protein particles according to the present invention is obtained. These reflect and contain the membrane proteome and lipidome of the unpurified membrane vesicles used as starting materials in step a), i.e., the whole or part, preferably a substantial part, of the membrane proteome and lipidome of the cells or organelles from which the unpurified membrane vesicles were prepared. The library of sarp protein particles is directly useful in itself, for example, in life science research, systems biology, etc., for testing the membrane proteome and / or lipidome of a given cell or organelle, in drug development, particularly in the drug screening process, in the development of antibodies, and in various pharmaceutical or cosmetic applications.

[0187] However, the library obtained by the method of the present invention is also particularly useful for acting as a starting material from which specific selected or specific types of sarp protein particles can be purified.

[0188] Accordingly, the present invention also provides a process for preparing purified saposin-lipoprotein particles, comprising the step of preparing a library according to the above process, and f) a further step of purifying at least one type of saposin-lipoprotein particle from said library.

[0189] The purification of at least one type of sarp protein particle in step f) can be carried out by any purification, extraction or separation method, preferably by the methods described herein.

[0190] The purification of at least one type of sarp protein particle from the library may be carried out, but is not limited to, by affinity purification including affinity chromatography and / or immunopurification, particularly by using an antigen or tag on a membrane protein present in the particle to be purified. In addition to or instead of this, the purification may be carried out by chromatography, particularly size exclusion chromatography; ultracentrifugation; dialysis; contact with biobeads that bind to surfactants; or the use of a concentrator.

[0191] Preferably, the purification of at least one type of salipro particle in step f) is carried out by affinity purification such as affinity chromatography. Preferably, the antigen or tag (also referred to herein as an "affinity tag") is present on a membrane protein or lipid present in at least one type of salipro particle to be purified. Preferably, the antigen or tag is present on a membrane protein in at least one type of salipro particle to be purified. However, the tag or antigen used for purification may be present on a lipid-linked polypeptide in the salipro particle. In this manner, the entire library is affinity purified in step f).

[0192] Preferably, the recognition moiety is used for affinity purification and the recognition moiety binds to an antigen or affinity tag present in the salipro particle.

[0193] As is known to those skilled in the art, affinity purification involves an antigen or affinity tag on the component to be purified (e.g., salipro particle) and the corresponding recognition moiety (e.g., an antibody in the case of an antigen or an Ni-NTA moiety in the case of a His-tag) used to generate the component from the mixture. Basically, various antigen or affinity tag / recognition moiety pairs known to those skilled in the art from protein affinity purification techniques can be used to purify at least one type of salipro particle according to the present invention in step f).

[0194] The interaction of an affinity tag / recognition moiety pair was developed to assist in protein purification and immobilization. Proteins may be modified at the genetic level using a specific peptide sequence that binds to a known recognition moiety, known as an affinity tag. Affinity tags are generally classified into three categories: a) a peptide sequence that binds to a small molecule, b) a fusion protein that binds to a small molecule, and c) a peptide tag or fusion protein that binds to an antibody. The affinity tag may be a small molecule having a simple binding pair. The affinity tag may covalently bind to a target protein, peptide, or lipid present in the unpurified membrane vesicles used in step a) of the method of the present invention. In this manner, the affinity-tagged sariplo particles may be immobilized on a matrix or resin that generates a binding pair or recognition moiety for the affinity tag. For example, nitrilotriacetic acid forms a complex with Ni 2+ (NTA-Ni 2+ ), defines a recognition moiety that binds to a protein modified with a histidine extension known as a histidine tag, and defines an affinity tag.

[0195] An "affinity tag" is given its ordinary meaning in the art. An affinity tag is any biological or chemical material that can be readily attached to a target biological or chemical material. The affinity tag may be attached to a target biological or chemical molecule by any suitable method. For example, in some embodiments, the affinity tag may be attached to the target molecule by genetic means. For example, a nucleic acid sequence encoding the affinity tag may be inserted near a sequence encoding a biological molecule present in an unpurified membrane vesicle. The sequence may be located anywhere (e.g., within, adjacent to, or near) in the nucleic acid that can express the affinity tag together with the biological molecule. In other embodiments, the affinity tag may be attached to the target biological or chemical molecule after the target biological or chemical molecule has been produced (e.g., expressed or synthesized). As an example, an affinity tag (e.g., biotin) may be chemically coupled (e.g., covalently) to a target protein or peptide to facilitate the binding of this target to streptavidin.

[0196] Examples of affinity tags include, for example, tags that bind to metals, such as histidine tags, GST (in glutathione / GST binding), streptavidin (in biotin / streptavidin binding). Other affinity tags include Myc or Max within the Myc / Max pair, or polyamino acids, such as polyhistidine. At various places in this specification, specific affinity tags are described in relation to binding interactions. The molecule with which the affinity tag interacts (e.g., binds) may be a known biological or chemical binding pair and is the "recognition moiety". It should be understood that the present invention includes, in any embodiment using an affinity tag, a series of individual embodiments each including the selection of any of the affinity tags described herein.

[0197] The recognition moiety may be any chemical or biological material that can bind to an affinity tag. The recognition moiety may be, for example, a small molecule, such as maltose (which binds to MBP, i.e., maltose-binding protein), glutathione, NTA / Ni2+, biotin (which can bind to streptavidin), or an antibody. The affinity tag / recognition moiety interaction will facilitate the connection of the target molecule to another biological or chemical material, or a substrate. Examples of affinity tag / recognition moiety interactions include polyhistidine / NTA / Ni2+, glutathione S-transferase / glutathione, maltose-binding protein / maltose, streptavidin / biotin, biotin / streptavidin, antigen (or antibody fragment) / antibody (or antibody fragment), etc.

[0198] Pairs of affinity tags or antigen / recognition moieties useful in step f) of the present invention are, for example, antibody / peptide interaction, antibody / antigen interaction, antibody fragment / antigen interaction, nucleic acid / nucleic acid interaction, protein / nucleic acid interaction, peptide / peptide interaction, protein / protein interaction, small molecule / protein interaction, glutathione / GST interaction, maltose / protein interaction that binds to maltose, carbohydrate / protein interaction, carbohydrate derivative protein interaction, peptide tag / metal ion-metal chelate interaction, peptide / NTA-Ni interaction, epitope tag (e.g., V5-tag, Myc-tag, FLAG-tag or HA-tag) / antibody interaction, protein A / antibody interaction, protein G / antibody interaction, protein L / antibody interaction, fluorescent protein (e.g., GFP) / antibody interaction, Fc receptor / antibody interaction, biotin / avidin interaction, biotin / streptavidin interaction, zinc finger / nucleic acid interaction, small molecule / peptide interaction, small molecule / target interaction and metal ion / chelating agent / polyamino acid interaction.

[0199] Any of the above affinity tags may be present on a membrane protein, lipid, or lipid-linked polypeptide in the suripro particles of the present invention, particularly in the types of suripro particles purified in step f). To purify the suripro particles in step f), any of the above recognition moieties may be used.

[0200] In certain embodiments of the invention, the tag is a fluorescent tag. In particular, a suripro particle obtained by the method of the present invention, or a single type of suripro particle present in a suripro particle library, bears a fluorescent tag. In preferred embodiments, a lipid, membrane protein, or lipid-linked polypeptide present in the suripro particle bears a fluorescent tag. Such fluorescent tags are useful for detecting and following a particular membrane protein or a particular type (species) of suripro particle during its production (optionally purification) in the method of the present invention. GFP and its variants are the most commonly used fluorescent tags. In addition to this, from actual tests, it has been found that membrane proteins can be specifically tagged in unpurified membranes or unpurified membrane vesicles, and then this is used in step a) for preparing a library according to the present invention. This tagging preferentially occurs at the gene level, for example, by genetic manipulation or by inserting a vector having a tagged transgene into the cells or organelles that are the source of the unpurified membrane vesicles.

[0201] Using the above techniques, the present invention provides a process for preparing suripro particles comprising a specific membrane protein of interest having an antigen or an affinity tag.

[0202] This procedure is exemplified below for an unpurified cell membrane containing a GFP fusion of the membrane protein GLUT5, but the invention is not limited thereto. Briefly, an unpurified membrane fraction containing unpurified membrane vesicles containing GFP-GLUT5 (step a) of the process according to the invention) is contacted with saposin A in a liquid environment (with or without a surfactant) (step b) of the process according to the invention and partially already step c)), and then surfactant micelles are removed using gel filtration chromatography / size exclusion chromatography in a surfactant-free buffer (step c) of the process according to the invention), thereby self-assembling the hydrophobic portion of the initial mixture into sariplonano particles. Due to the complexity of the unpurified membrane used as the starting material, this process yields a heterogeneous library of sariplo particles containing a large number of different membrane proteins. Some of the sariplo particles in the library contain the fluorescently labeled GFP-GLUT5 membrane protein, as shown in Figure 4b below, other sariplo particles contain other membrane proteins, and some sariplo particles contain no membrane proteins and only membrane lipids from the unpurified membrane.

[0203] In one embodiment, the process according to the invention provides a library of sariplo particles, wherein each sariplo particle consists essentially of at least one lipid-bound polypeptide and a component of the membrane of an unpurified cell or organelle, and in particular, each sariplo particle consists essentially of at least one lipid-bound polypeptide, a membrane lipid from the membrane of an unpurified cell or organelle, and optionally, a membrane protein.

[0204] As used herein, the term "essentially" means that trace amounts of additional components used in the processes according to the invention, such as components of the crude membranes or agents (e.g., surfactants) used in this process, may also be present in the saposin-like particles. The saposin-like particles essentially consist of at least one lipid-bound polypeptide and components of the membrane obtained from the membranes of cells or organelles, but in particular, it means that no additional lipids and / or proteins are added. In particular, no additional synthetic, purified, and / or exogenous lipids and / or proteins are added to the processes according to the invention.

[0205] In a preferred embodiment of the invention, no additional lipids other than the lipid components of the crude membrane vesicles are added to the processes according to the invention. In an even more preferred embodiment of the invention, no additional membrane proteins other than the protein components of the crude membrane vesicles are added to the processes according to the invention.

[0206] The present invention provides libraries of saposin-lipoprotein particles and / or saposin-like particles that can be obtained according to any of the above-described processes.

[0207] The saposin-like particles that can be obtained by the processes according to the invention and are included in the library of saposin-like particles of the present invention differ from the particles of the prior art in a plurality of characteristics. For example, these particles contain components of the membranes of crude cells and organelles. In particular, these particles contain membrane proteins from the membranes of cells and organelles, especially membrane proteins that are still preserved in their natural cell or organelle membrane lipid content. In a preferred embodiment, the saposin-like particles of the present invention contain only membrane lipids as the membrane protein component and the lipid component, and optionally contain membrane proteins from one specific type of cell or organelle membrane. In a particularly preferred embodiment, the saposin-like particles of the present invention contain only membrane proteins and lipids derived from the membranes of the same cell or organelle as the membrane protein component and the lipid component, if present.

[0208] In addition, the saposin particles of the present invention are characterized by their inherent size flexibility and the ability to adapt to the respective sizes of the membrane components incorporated into the lipoprotein particles. Thereby, a wide variety of membrane proteins, their complexes, membrane domains or membrane components can be incorporated into the saposin particles of the library. Thus, the library of the present invention is heterogeneous not only in terms of the content of each individual saposin particle, but also in terms of the size of each individual saposin particle. "Empty", i.e., lipid-only saposin particles, will be smaller than those containing a given membrane protein, and those containing a given membrane protein will be smaller than those containing a multimeric membrane protein complex. This size flexibility allows the saposin particle library to capture an unbiased array of the membrane proteome and lipidome of a given cell or organelle in its native content.

[0209] Typically, the particles in the saposin particle library differ in terms of their protein composition. These particles may also differ in terms of their lipid composition, which reflects the different lipid compositions of the membrane domains in the membrane of the cell or organelle used as the starting material. Advantageously, the library obtained according to the present invention may provide particles containing a part or component of the membrane. Another advantage is that the native environment of the membrane lipids and, optionally, the membrane proteins is maintained in the saposin particles of the present invention.

[0210] A library of saposin particles or saposin particles obtainable according to the method of the present invention is also provided for use in medicine, particularly for use in preventing, treating or reducing the severity of a disease, or for use in diagnostic methods, cosmetic treatments, or as a vaccine formulation.

[0211] For example, the saposin particles of the present invention may be included in a pharmaceutical composition for delivering one or more membrane proteins and / or lipids to an individual in need of treatment, wherein the composition comprises the particles of the present invention as described above.

[0212] In addition to the particles of the present invention, the pharmaceutical composition may optionally (further) contain a pharmaceutically acceptable vehicle, carrier or adjuvant. When the particles of the present invention are for use in a pharmaceutical composition, the particles and the individual components of the pharmaceutical composition should be pharmaceutically acceptable. As used herein, the term "pharmaceutically acceptable" refers to components, compounds or agents that, within the scope of sound medical judgment, do not cause excessive toxicity, irritation, allergic reactions, etc., are compatible with a reasonable benefit / risk ratio, and are suitable for use in contact with the tissues of humans and lower animals.

[0213] In yet another aspect, the present invention provides a method of treating an individual in need of treatment with a therapeutically effective amount of the pharmaceutical composition described above. A "therapeutically effective amount" of a pharmaceutical composition, as used herein, is an amount effective to treat or reduce the severity of a disease or condition being treated. The term "individual", as used herein, means an animal, such as a mammal, and more particularly a human.

[0214] The pharmaceutical composition of the present invention may be administered to humans and other animals orally, rectally, parenterally, intracapsularly, intravaginally, intraperitoneally, topically (such as by powder, ointment or droplet), orally, by aerosol, oral or nasal spray, etc., depending on the severity of the disease or condition being treated. In particular, the pharmaceutical composition may be formulated for enteral administration, parenteral administration and / or topical administration. The pharmaceutical composition may be administered as a capsule, injection or infusion, a brushable composition or a drinkable composition, or as an aerosol. In some embodiments of the pharmaceutical composition of the present invention, the particles of the present invention are present in solid form, as a suspension, or in solution.

[0215] The particles of the present invention are also useful in diagnostic applications and / or cosmetic applications. For example, salipro particles containing a detectable antigen or tag (such as those described above) may be used as a diagnostic agent and applied for diagnostic purposes. The antigen or tag itself may be included in a membrane protein present in the salipro particles, or may be made by a membrane protein, but these may be connected to the lipid-binding polypeptide or lipid component of the particle. Examples of the diagnostic agents and tools for life science research according to the present invention include particles having a tagged and incorporated membrane protein, a tagged lipid-binding polypeptide, a tagged lipid, an incorporated fluorophore or contrast agent (for example, for MR imaging). The tag may be, for example, a fluorescent tag.

[0216] In another aspect, the particles of the present invention are useful as a vaccination preparation, as a carrier thereof, or as a drug delivery vehicle. Many pathogen antigens that can be particularly potent in vaccination are exposed on the surface of eukaryotic or prokaryotic pathogens or diseased cells (for example, cancer cells) in a patient and / or are included in the outer cell membrane. These antigens may be derived from, for example, pathogen lipids, other hydrophobic biomolecules or membrane proteins. The term pathogen, as used herein, does not include viruses. Using the particles of the present invention, such antigens can be effectively incorporated into the particles and then used as an antigen-presenting delivery vehicle in a vaccination preparation. Thus, the particles of the present invention are also useful for functioning as an antigen-presenting delivery vehicle for generating antibodies against lipids or membrane proteins in a suitable host animal, preferably in mammals (for example, rabbits, goats, llamas, mice and primates).

[0217] Furthermore, the present invention provides, in a further aspect, the use of the salipro particles of the present invention or a library of the salipro particles of the present invention as tools for drug development, drug screening, drug discovery.

[0218] For example, a specific membrane protein drug target, such as a cell surface receptor or an ion channel, may be incorporated into the particles of the present invention and may be solubilized in its native state. The particles may then be purified from the library described above. Such particles may then be used in an assay to test the activity of the drug target membrane protein in its native lipid bilayer environment, or may be used in drug screening to identify new drugs.

[0219] Conversely, to identify a novel molecular drug target, such as a membrane protein or lipid present in the membrane of a target cell or organelle, the entire library of sariplo particles obtained from a specific cell or organelle that may be a disease target may be used for drug screening purposes.

[0220] The libraries and methods described herein can be used as tools for membrane protein purification, for membrane protein expression, for membrane and / or membrane protein research, particularly for lipidomics and proteomics, preferably for the isolation, identification and / or testing of membranes and / or membrane proteins, or for the creation of lipidome or proteome libraries or databases.

[0221] Furthermore, the particles of the present invention, whether purified or as part of the entire library, may be immobilized on a solid support to be useful for applications such as surface plasmon resonance (SPR) or for biosensor applications.

[0222] The particles of the present invention are generally useful for solubilizing insoluble membrane proteins and membrane domains or components into an aqueous solution in their native membrane bilayer microenvironment. Thus, the present invention provides a variety of new applications in membrane protein research. For example, with the particles of the present invention, membrane proteins incorporated into the particles of the present invention can be tested by methods such as nuclear magnetic resonance (NMR), X-ray crystallography, electron microscopy (EM), mass spectrometry, isothermal titration calorimetry (ITC), differential light scattering, small angle X-ray scattering (SAXS).

[0223] The library of the present invention is particularly useful for research in the field of systems biology, particularly in the fields of lipidomics and membrane proteomics. The library of the present invention would be suitable for capturing and solubilizing the lipidome or proteome of cells or cell organelles. Typical analytical techniques in lipidomics and proteomics are techniques such as mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy, fluorescence spectroscopy, and computer calculation methods. Applying these methods or techniques to the sarpoparticles of the present invention enables further elucidation of the roles of natural lipids and membrane proteins in many metabolic diseases (e.g., cancer, autoimmune diseases, obesity, atherosclerosis, stroke, hypertension, and diabetes).

[0224] The advantage of incorporating unpurified membranes into the particles of the library of the present invention is that it represents a snapshot of the microdomains and components of the actual membrane as the source. In contrast, typical saposin lipids containing particles known from the prior art are composed of a synthetic or heterogeneous mixture of purified lipids and optionally proteins, and the lipids are usually derived from a source completely different from the membrane proteins present in the particles.

Brief Description of the Drawings

[0225] The present invention will be described below with reference to the drawings showing specific embodiments of the present invention. However, the present invention is defined in the claims and generally described herein. The present invention should not be limited to the embodiments shown for the purpose of explanation in the following drawings.

[0226]

Figure 1

[0227]

Figure 2

[0228]

Figure 3

[0229]

Figure 4

[0230]

Figure 5

[0231]

Figure 6

[0232]

Figure 7

[0233]

Figure 8

[0234]

Figure 9

[0235]

Figure 10

[0236]

Figure 11

[0237]

Figure 12

[0238] Figure 1a) shows a prior art apolipoprotein A-1 (see, for example, the above-mentioned European Patent No. 1596828) comprising a nanodisc particle A containing lipid B and apolipoprotein A-1 as a lipid-binding polypeptide C. In contrast to the nanodiscs derived from prior art apolipoproteins, the lipid-binding polypeptides of the present invention do not surround the lipid in a double-belt-like fashion (see C in Figure 1a). Rather, the particles of the present invention are held together by a core containing membrane lipids surrounded by two or more lipid-binding polypeptides of substantially V-shaped or boomerang-shaped configurations arranged in a head-to-tail arrangement, and there is substantially no direct protein-protein contact between the individual lipid-binding polypeptides within a given particle of the present invention (see Figures 2a to 2f).

[0239] Figure 1b is a schematic diagram of the process used to create the saposin lipoprotein particle G of the above prior art (for example, International Publication No. 2014 / 095576). Here, SAPLIP D is solubilized with purified lipid E and surfactant F and incubated with purified membrane protein 4a to generate particle G. Lipid E is purified and derived from a source different from membrane protein 4a. Membrane protein 4a does not exist in a membrane or vesicle and is in a state solubilized by an artificial surfactant (lipid E and surfactant molecule F binding to the hydrophobic surface of membrane protein 4a in the center of Figure 1b). Thus, in particle G, membrane protein 4a is not embedded in its native membrane environment but rather in a mixture containing artificial or exogenous lipid E and, possibly, surfactant F as well.

[0240] Figures 2a) to 2f) are schematic diagrams of saporin particles obtained according to a particular embodiment of the present invention. Particles 1a, 1b, and 1c comprise a plurality of different membrane lipids 3 and optionally membrane proteins 4a, 4b. The membrane lipids 3 and the membrane proteins 4a and 4b are derived from the same cell or organelle membrane used to prepare the particles. The lipids 3 are neither uniform nor homogeneous but differ from one another as is typical in biological membranes. In addition, depending on the source from which the unpurified membrane vesicles 5 are obtained (see FIGS. 3a and 3b), the composition of the membrane varies, and thus a mixture of lipids 3 and optionally membrane proteins is present in the saporin particles. In a further embodiment, although not shown, the saporin particles may also contain additional components typically present in cell or organelle membranes.

[0241] Particles 1a - 1c are not drawn to scale. Depending on the size of the membrane proteins 4a, 4b incorporated into particle 1a or 1b, the lipid - only particle 1c may be significantly different in size compared to the other particles 1a, 1b. Also, particles 1a and 1b may differ in size, lipid, and optimally in the composition of the membrane proteins. Also, particle 1c may differ in terms of size, for example, if part of a lipid raft is incorporated into the saporin particle as a whole. Note that the saporin particles of the present invention can be flexibly varied in size. For example, particle 1b carrying an oligomeric membrane protein is larger and contains more saporin subunits 2 compared to particle 1a containing a monomeric membrane protein.

[0242] Figures 2a) and 2b) show, in a simplified schematic form, a saporin particle 1c comprising SAPLIP as a lipid - binding polypeptide 2 and cell or organelle membrane lipids 3. a) is shown as a side view and b) is shown as a top view.

[0243] Figures 2c) and 2d) show the sarply particles 1a and are different from 1c in that they further contain the membrane protein 4a. c) is shown as a side view and d) is shown as a top view. The membrane protein 4a may be an integral transmembrane protein in monomeric form. However, it may also be an oligomeric or surface membrane protein, a bidirectional protein in a state bound to a lipid, a protein immobilized on a lipid, or a chimeric protein having a fused hydrophobic and / or transmembrane domain as shown in Figures 2e) or 2f), and all of these may be in monomeric or oligomeric states.

[0244] Figures 2e) and 2f) show the sarply particles 1b and are different from 1c in that they further contain the oligomeric membrane protein 4b. e) is shown as a side view and f) is shown as a top view. The particle 1b shows flexibility in size and adapts to the size of the oligomeric membrane protein 4b incorporated therein. In the embodiment shown in Figures 2e) or 2f), the particle 1b contains 3 SAPLIP molecules 2 per particle arranged in a head-to-tail manner. The hydrodynamic radius of the particle containing 3 SAPLIP molecules is in the range of 5 to 20 nm depending on the hydrophobic drug incorporated therein.

[0245] Figures 3a) and 3b) again show, in simplified and schematic form, the process according to the invention for preparing the sarply particle library 7 from the unpurified membrane vesicles 5. In Figure 3b), the library 7 is schematically shown as a mixture of representative sarply particles 1a - 1c that differ in their membrane lipids 3 and, optionally, the proteins cargo 4a, 4b and / or size. Of course, in reality, the library 7 will contain thousands of different sarply particles. Similarly, the starting mixture 6 will contain a large number of different unpurified membrane vesicles.

[0246] As shown in Fig. 3a), the particles 1a - 1c of the present invention are prepared by mixing purified SAPLIP2 with unpurified membrane vesicles 5, 5' and allowing the particles 1 to self - assemble. The composition 6 containing the unpurified membrane vesicles 5 and 5' may be an unpurified membrane fraction directly obtained after lysis of cells or cell organelles. Vesicles 5 and 5' usually form spontaneously upon lysis or disruption of the membrane. The composition 6 of the starting material is shown schematically only as containing representative vesicles 5 and 5', one of which (5') may be small and contain only membrane lipids, and the other (5) may be large and contain, in addition to membrane lipid 3, membrane proteins 4a and 4b. The unpurified membrane vesicles used as the starting material 6 of the process of the present invention may be very heterogeneous in terms of size and content. A homogenization or specific vesicle - preparation step is not necessarily required, but may be present. Of course, a composition containing even more homogeneous unpurified membrane vesicles may also be used as the starting material 6.

[0247] Fig. 3a) shows the preparation of the library 7 shown in Fig. 3b). The particles 1a - 1c of the present invention are prepared by mixing purified SAPLIP2 with unpurified membrane vesicles 5, 5' containing both membrane lipid 3 derived from unpurified membranes and optionally one or more membrane proteins 4a and / or 4b. Then, the self - assembly X of the particles 1 occurs, for example, at a pH of about 5.0 to about 10.0. The unpurified membrane vesicles 5, 5' may optionally contain surfactant molecules or be associated with surfactant molecules (not shown herein). The same will hold true for the saposin molecules 2. The membrane lipid 3 associated with the membrane proteins 4a and / or 4b is preferably transported out of the natural lipid environment of the membrane proteins into the membrane of cells or cell organelles exclusively before providing the unpurified membrane vesicles 5, 5'. In particles 1a and 1b, the respective membrane proteins 4a and 4b are embedded in the components of the hydrophobic part of the membrane from which they are supplied. Preferably, the membrane proteins are in the same or a similar conformation while bound to their native membrane.

[0248] Figure 3b) shows a library of sariplo particles containing representative particles 1a, 1b, and 1c. Particles 1a and 1b contain membrane proteins 4a, 4b. Particles 1a - 1c (also described in more detail in Figures 2a) - 2f), shown as specific embodiments of the present invention) are approximately disk - shaped, having a flat disk - like lipid bilayer that ranges from approximately circular to square - shaped, and is surrounded by the amphiphilic α - helices of two or three SAPLIP molecules 2. The lipids 3 of the unpurified membrane vesicles 5, 5' aggregate to form a disk - bilayer - like structure with a distinct size inside particles 1a - 1c. SAPLIP 2 defines the boundary of the disk - shaped bilayer of particles 1a - 1c, and the inside thereof is hydrophobic, that is, composed of lipid aliphatic acyl chains and lacks a hydrophilic or aqueous core. Particles 1a - 1c are held together mainly by the hydrophobic interaction between the lipids 3 of the unpurified membrane vesicles 5 within the bilayer core of particles 1a - 1c, and the hydrophobic interaction between the lipids 3 of the unpurified membrane vesicles 5 and the hydrophobic sites of the amphiphilic helices of SAPLIP 2 facing the inside of the particles. In its smallest form, particle 1c is thought to contain two SAPLIP molecules 2 and at least about 2 - 5 lipid molecules 3 of the unpurified membrane vesicles 5, 5'. However, the particles 1a - 1c of the present invention can be flexibly sized. Depending on the size of the cargo incorporated (e.g., lipid domains, membrane proteins, etc.) and the molar ratio of the components used in the preparation, multiple, i.e., more than two, SAPLIP 2s, many lipids 3 of the unpurified membrane vesicles 5, and optionally one or more membrane proteins 4a and / or 4b can be accommodated. For example, the particles may contain 2 - 20, particularly 2 - 10, SAPLIP 2s and optionally one or more membrane proteins. Depending on the size of the membrane proteins 4a, 4b incorporated into particles 1a, 1b, the particles may be significantly larger than lipid - only particle 1c. Generally, the increase in particle size is also reflected by the number of SAPLIP 2s per particle, and this number may be more than two. The particles of the present invention may contain, for example, 2 - 20, particularly 2 - 10, SAPLIP molecules 2.

[0249] Figures 4a) and 4b) are basically the same as Figures 3a) and 3b), except that the membrane protein 4c having the affinity tag 8 is included in the unpurified membrane vesicles 5 of the starting material 6. As a result of the self-assembly Y after contacting the unpurified membrane vesicles 5, 5' with the purified SAPLIP2, the resulting library 7' contains the particles 1d containing the membrane protein 4c and the affinity tag 8. The descriptions of Figures 3a) and 3b) above are equally applicable to Figures 4a) and 4b).

[0250] Figure 5 is a schematic diagram of one embodiment of a process according to the invention for purifying specific particles from a library of the invention by performing step f) as generally further described above. As the starting material, the library 7' is used, which can be obtained as shown in Figures 4a) and 4b) above. Affinity purification is performed on the library 7', for example, using an affinity column 9. The column 9 contains a recognition moiety that binds to the affinity tag 8. For example, when the affinity tag 8 is a His-tag, the recognition moiety of the affinity column 9 is Ni-Nta. Although purification in column form is shown in Figure 5, a batch purification process is also possible.

[0251] By passing library 7' through affinity column 9, specific binding of particle 1d to the recognition portion of column 9 occurs via affinity tag 8. Particle 1b and 1c, and other components of the library or test pieces do not bind to or only non-specifically bind to affinity column 9. Thus, the resulting flow-through fraction 1.) essentially does not contain particle 1d. Some of the remaining components of the library (e.g., particles 1b and 1c) may non-specifically bind to affinity column 9, but are removed by performing at least one washing step 2.). Finally, the purified particle 1d can be eluted by an elution step 3.) by disrupting the binding of affinity tag 8 to the recognition portion of affinity column 9. Such disruption can be performed by a high-concentration salt wash solution, by enzymatic cleavage, or, in the case of the His-tag / Ni-NTA tag / recognition portion pair, using imidazole. The elution fraction obtained after 3.) contains the particle 1d of the present invention in a purified form.

Example

[0252] The following examples are specifically useful for further explaining the present invention with reference to specific embodiments and drawings, but the specific embodiments and drawings are not intended to limit the present disclosure.

[0253] The following abbreviations are used. GF buffer (pH 7.5): 20 mM HEPES (pH 7.4) and 150 mM NaCl GFP Green fluorescent protein Glut5: Transporter, membrane protein HEPES: 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid His Histidine HN buffer: 20 mM HEPES, 150 mM NaCl, pH 7.4 HN-D buffer HN buffer containing 0.2% DDM M: Molar concentration RT: Room temperature SEC: Size Exclusion Chromatography TCEP: Tris(2-carboxyethyl)phosphine TEV: Tobacco Etch Virus Tris: Tris(hydroxymethyl)aminomethane

[0254] The purified saposin A used in the following experiment was prepared as follows. Using a vector having the coding region of human saposin A (SEQ ID NO: 1) inserted into the pNIC-Bsa4 plasmid, transformed, and expressed in the E. coli Rosetta gami-2(DE3) (Novagen) strain, the expression of saposin A protein was carried out. The cells were grown at 37 °C in TB medium supplemented with tetracycline, chloramphenicol, and kanamycin and induced with 0.7 mM IPTG. Three hours after incubation, the cells were collected by centrifugation at 12,000×g for 15 minutes. The supernatant was discarded, and the cell pellet was resuspended again with lysis buffer (20 mM Hepes, pH 7.5, 150 mM NaCl, 20 mM imidazole) and disrupted by sonication. The lysate was centrifuged at 26,000×g for 30 minutes, the supernatant was heated to 85 °C over 10 minutes, and then further centrifuged at 26,000×g for 30 minutes. Fractional IMAC purification was performed by inverted rotation using Ni Sepharose™ 6 Fast Flow medium for 60 minutes by batch adsorption of the supernatant. After binding saposin A to the IMAC resin, the chromatography medium was placed in an open gravity flow column with an inner diameter of 10 mm, and unbound proteins were removed by washing with 15 column volumes of lysis buffer. The resin was washed with 15 column volumes of wash buffer WB2 (20 mM Hepes pH 7.5, 150 mM NaCl, 40 mM imidazole). Saposin A was eluted by adding 5 column volumes of elution buffer EB (20 mM Hepes pH 7.5, 150 mM NaCl, 400 mM imidazole). The eluate was dialyzed overnight against gel filtration buffer GF, pH 7.5 (20 mM Hepes pH 7.5, 150 mM NaCl) supplemented with recombinant TEV protease. TEV protease containing a non-cleavable His-tag was removed from the eluate by flowing it over 2 ml of IMAC resin.The cleaved target protein was concentrated using a centrifugal filter unit to a volume of 5 ml and loaded onto a HiLoad Superdex™ 200 16 / 60 GL column using an ÄKTAexplorer™ 10 chromatography system (both from GE Healthcare). The peak fractions were pooled and concentrated to a protein concentration of 1.2 mg / ml. This protein sample was snap-frozen in liquid nitrogen and stored at -80 °C.

[0255] Example 1a The unpurified yeast cell membrane fraction was obtained from yeast cells expressing GFP-GLUT5. The membrane fraction contained naturally occurring unpurified membrane vesicles, which were incubated with a surfactant and saposin A, and then the surfactant was removed in a surfactant-free buffer using gel filtration chromatography / size exclusion chromatography (SEC). This caused the membrane components present in the initial mixture to self-assemble into a library of nanoscale saposin particles. In particular, monodisperse saposin particles containing membrane lipids and GFP-GLUT5 could be identified within this library.

[0256] 1. Membrane preparation Unpurified yeast membranes were obtained from yeast cells expressing rat GLUT5 from the GAL1-inducible TEV-cleavable GFP-His8 2μ vector pDDGFP2 known in the prior art. The vector was transformed into the S. cerevisiae strain FGY217 (MATa, ura3-52, lys2Δ201 and pep4Δ), and then GFP-GLUT5 was overexpressed in its cell membrane.

[0257] To create the crude membrane, cells were harvested from 12 L of S. cerevisiae culture, resuspended in a buffer containing 50 mM Tris-HCl pH 7.6, 1 mM EDTA, and 0.6 M sorbitol, and lysed by mechanical disruption. Membranes were isolated by ultracentrifugation at 195,000 g for 3 hours, homogenized in 20 mM Tris-HCl pH 7.5, 0.3 M sucrose, 0.1 mM CaCl2, frozen in liquid nitrogen, and stored at -80 °C.

[0258] 2. Preparation of the salipro particle library In particular, 20 μl of crude yeast membranes containing naturally occurring crude membrane vesicles including GFP-GLUT5 were mixed with 60 μl of HN buffer (20 mM HEPES, 150 mM NaCl, pH 7.4) and 20 μl of HN buffer supplemented with 5% DDM, and then incubated at 4 °C for 1 hour. The membrane lysate was then washed by ultracentrifugation at 47 krpm (100,000 g) for 30 minutes using a TLA-55 rotor from debris and protein aggregates. Then, 5 μl of the washed membrane fraction was mixed with increasing amounts (5 - 10 - 20 - 30 - 40 μl) of saposin A (1.2 mg / ml, HN-buffer), incubated at 37 °C for 5 minutes to self-assemble the salipro particles. Only the lipids present during the setting are derived from the crude membrane and crude membrane vesicles.

[0259] Thereafter, the sample volume was adjusted to 50 ul with HN buffer and centrifuged at 13 krpm for 10 minutes. SEC analysis was performed as follows (using a Shimadzu HPLC system). A 35 μl sample was injected into a 5 / 150 Superose6 increasing column (GE Healthcare) at a flow rate of 0.3 ml / min, and the presence of the fluorescent GFP tag was monitored online. The SEC buffer consisted of HN buffer and did not contain surfactant.

[0260] As a negative control, saposin A was completely omitted from the experimental setup (0 ul SapA), the volume was adjusted to 50 μl using NH buffer, and then the samples were processed as previously described.

[0261] 3. Results These results are shown in Figure 6 and demonstrate that membrane proteins of membrane lipids, such as GFP-GLUT5 from unpurified cell membranes, can be incorporated into soluble saposin particles showing a monodisperse peak and stabilized. Increasing the amount of saposin improves the incorporation efficiency and the monodispersity of the peak. Thus, saposin A and GFP-GLUT5, which are lipids from unpurified membranes, associate in a manner that includes the incorporated membrane protein and forms water-soluble particles. Only GFP-GLUT5 is monitored by its fluorescence in SEC analysis, and the resulting library of saposin particles also contains a large number of other saposin particles that include the proteome and lipidome of the remaining membranes of the yeast cells that are the source of the unpurified membrane fraction. This can be confirmed, for example, by mass spectrometry, SDS-PAGE, and / or probing with antibodies that bind to other native yeast membrane proteins and lipids present in the resulting library of saposin particles.

[0262] As a negative control, in the absence of saposin from the setup, GFP-GLUT5 (and thus also the remaining membrane proteins) is not soluble and aggregates (see the high invalid peak).

[0263] Example 1b 1. Setup Unpurified yeast membranes containing unpurified membrane vesicles and rat GLUT5 were prepared as described in Example 1a.

[0264] 2. Process modifications for saposin particle library creation Two different methods for producing saposin-GFP-GLUT5 particles from unpurified membrane extracts were evaluated.

[0265] In the first method, 25 μl of saposin A (0.75 mg / ml) was added to 1 μl of the crude membrane extract containing crude membrane vesicles and GFP-GLUT5, and incubated at 37 °C for 5 minutes. Then, 24 μl of HN-D buffer was added to the above mixture, and incubated at 37 °C for an additional 5 minutes. Next, the sample was centrifuged at 13 krpm for 10 minutes, and SEC analysis was performed as in Example 1a.

[0266] In the second method, 1 μl of the crude membrane extract containing crude membrane vesicles and GFP-GLUT5 was first added by adding 24 μl of HN-D buffer, and then incubated at 37 °C for 5 minutes. Next, this sample was centrifuged at 13 krpm for 10 minutes. The lysate suspension was collected, 25 μl of saposin A (0.75 mg / ml) was added, and incubated at 37 °C for an additional 5 minutes. Then, as described in Example 1a, the sample was analyzed by SEC.

[0267] 3. Results The data shown in Figure 7 indicate that both methods function independently to obtain a library of salipro particles containing soluble salipro-GFP-GLUT5 particles when saposin A is added either before or after incubating the crude membrane with HN-D buffer at 37 °C for 5 minutes. In both cases, the native lipids of the crude membrane are still present. Interestingly, when saposin A is added directly to the crude membrane, only very slight protein aggregation is observed in a non-effective volume. Importantly, both methods work well, thereby giving the process a certain flexibility when reconstituting a library of salipro particles from a complex crude membrane extract.

[0268] Example 2 1. Setup A crude yeast membrane containing rat GFP-GLUT5 and crude membrane vesicles was prepared as described in Example 1a.

[0269] 2. Salipro generation, titration 20 μl of crude yeast membranes were mixed with 20 μl of HN buffer supplemented with 60 μl of HN buffer and 5% DDM at 4 °C and solubilized over 1 hour. The membrane lysate was then washed from debris and protein aggregates by ultracentrifugation using a TLA-55 rotor at 47 krpm (100,000 g) for 30 minutes. Next, 5 μl of the washed membrane lysate containing crude membrane vesicles was mixed with different volumes (12, 20, 30, and 40 μl) of saposin A (4 mg / ml, HN buffer) and incubated at 37 °C for 5 minutes. Subsequently, the sample volume was adjusted to 50 μl using HN buffer and centrifuged at 13 krpm for 10 minutes. SEC analysis (using a Shimadzu HPLC system) was performed by injecting 35 μl of the sample onto a 5 / 150 Superdex 200 Increase column (GE healthcare) at a flow rate of 0.3 ml / min and monitoring the presence of the fluorescent GFP tag online. Again, SEC was performed in the absence of surfactant using HN buffer to facilitate the reconstitution of membrane proteins into saposin particles.

[0270] As a control, 5 μl of the washed membrane lysate was mixed with 45 μl of NH buffer supplemented with 0.03% DDM and centrifuged at 13 krpm for 10 minutes. SEC analysis (using a Shimadzu HPLC system) was performed in HN buffer containing 0.03% DDM by injecting 35 μl of the sample onto a 5 / 150 Superdex 200 Increase column (GE healthcare) at a flow rate of 0.3 ml / min and monitoring the presence of the fluorescent GFP tag online. The purpose of this control sample (yield control sample) was to serve as a reference point for determining the amount of GFP-GLUT5 solubilizable under conditions where surfactant is permanently present, relative to the yield of GFP-GLUT5 reconstituted into saposin particles in a surfactant-free buffer system.

[0271] 3. Results The results shown in Figure 8 indicate that increasing the amount of saposin A improves the amount of GFP-GLUT5 reconstituted into saposin particles of the library.

[0272] To quantify the reconstruction yield, the ratio of the GFP-GLUT5 peak value from the "yield control sample" was compared with the peak value of the reconstructed sample. As a result, it was shown that 36% of GFP-GLUT5 was reconstructed for the 12 μl SapA sample, 57% for the 20 μl SapA sample, 65% for the 30 μl SapA sample, and 74% for the 40 μl SapA sample. In summary, this indicates that increasing the amount of saposin can increase the incorporation of membrane proteins from the crude membrane into the saposin particles.

[0273] Example 3: The solubility of the saposin membrane protein components of the library was analyzed.

[0274] 1. Background The crude membrane contains a large number of different membrane proteins. In the process according to the present invention, not only the fluorescently labeled GFP-GLUT5 but also various other yeast membrane proteins from the crude membrane are incorporated into the saposin particles. In the absence of a surfactant, the membrane protein fraction is insoluble and aggregates in a surfactant-free buffer system, generating a large invalid peak in SEC analysis. However, it was shown that the membrane proteins present in the saposin particle library remained soluble in a surfactant-free buffer system when embedded in the saposin particles.

[0275] 2. Setup In the same experimental setup as in Example 1a, based on UV absorbance at 280 nm instead of GFP fluorescence, the SEC signal was analyzed and focused (zoomed) on the invalid peak indicating the aggregated membrane proteins.

[0276] 3. Results In the absence of saposin, the membrane proteins from the crude membrane aggregate in a surfactant-free buffer system, as indicated by a large invalid peak that appears at approximately 4 minutes in SEC analysis (see Figure 9, 0 μl SapA).

[0277] In contrast, by increasing the amount of saposin added to the lysate, the amount of aggregated membrane proteins decreases accordingly. This indicates that due to the incorporation of the obtained membrane proteome library into the saposin particles, the membrane proteins remain soluble. In the sample containing the highest amount of saposin (Figure 9, 40 μl SapA), almost no protein aggregates were detected.

[0278] In summary, this data shows that after performing the method of the present invention, all membrane proteins from unpurified membrane vesicles remain soluble due to the successful reconstitution into the corresponding library of saposin nanoparticles even when the surfactant is removed in a surfactant-free environment. Therefore, it is possible to create a library of saposin particles-membrane protein / proteome and / or membrane lipid / lipidome derived from unpurified membranes.

[0279] Example 4: The solubility of the saposin membrane lipid components of the library was analyzed.

[0280] 1. Setup Perform different SEC analyses with the same experimental setup as in Example 1a (note the differences in the axis scales and axis intercepts in Figures 9 and 10), with a focus (zoom) on the peaks derived from monomeric saposin and saposin particles of only lipids.

[0281] 2. Results The results shown in Figure 10 indicate that when saposin A is added to the unpurified membrane, not only sariplo particles containing membrane proteins but also lipid-only (i.e., "empty") sariplo particles are generated. Increasing the amount of saposin (5, 10, 20, 30, and 40 μl) increases the generation of lipid-only sariplo particles. From SEC analysis, a peak (shown) derived from lipid-only sariplo particles appears at 7.6 minutes, while the corresponding peak for monomeric saposin ("free saposin") appears at 8.2 minutes (shown). This data indicates that lipids from unpurified membrane vesicles generate sariplo lipid-only particles at neutral pH.

[0282] As a negative control, one sample was analyzed in the absence of saposin (0 μl SapA).

[0283] This experiment using increasing amounts of saposin was performed on a Superose 6 5 / 150 column (GE Healthcare). Since the SEC separation of the peaks associated with these two saposins was not well separated, the experiment was repeated using a Superdex 200 5 / 150 column (GE Healthcare) (Figure 11).

[0284] One sample was prepared as described in Example 2 for the "12 ul SapA" sample (shown as "Sap + unpurified membrane" in Figure 11). At this time, SEC analysis was performed using UV absorbance at 280 nm. As a first control, one sample contained only saposin A to show the position of monomeric saposin without lipids in the SEC profile. The second control sample contained saposin A particles obtained by the method described in International Publication No. WO 2014 / 095576, i.e., by incubating saposin A with purified lipids.

[0285] The data presented herein clearly shows that both the cell membrane lipidome and proteome can be incorporated into sariplo particles to generate their respective libraries.

[0286] Example 5: In this example, a library of saposin particles was prepared as described in the above example, but only HN buffer without surfactant was used. This experiment also successfully incorporated GFP-GLUT5 from the crude membrane into the soluble saposin particles.

[0287] Example 6: Purification of Specific Saposin Particles from the Library In this example, a membrane protein library obtained according to Example 3 is prepared. The library of saposin particles essentially encompasses the entire yeast membrane proteome and also includes saposin particles containing GFP-GLUT5. The latter particles are purified by the TEV-cleavable GFP-His8 tag present in the GFP-Glut5 construct.

[0288] For this library, Ni-NTA affinity purification (e.g., Qiagen) is performed according to the manufacturer's instructions. After a predetermined washing step, the saposin particles containing GFP-GLUT5 are eluted by TEV protease cleavage or imidazole.

Claims

1. A process for preparing a library of saposin lipoproteins, wherein the library means a series of different saposin lipoproteins, including a heterogeneous mixture of saposin lipoproteins, said different saposin lipoproteins comprising different compositions of one or more membrane lipids and one or more membrane proteins, said particles comprising membrane components from cell or organelle membranes and a lipid-binding polypeptide or derivative thereof which is a saposin-like protein belonging to the SAPLIP family of proteins that interact with lipids, The lipid-binding polypeptide which is a saposin-like protein belonging to the SAPLIP family of proteins that interact with lipids is characterized by a conserved α-helical three-dimensional structure stabilized by a saposin folding moiety and highly conserved intramolecular disulfide bonds, The derivative has a saposin folding moiety and contains six cysteine residues corresponding to the six cysteines in the SAPLIP found in members of saposin A (SEQ ID NO: 1), and i) a protein having at least 90% sequence identity to the full-length sequence of SEQ ID NO: 1, 2, 3, 4, 5 or 6, said protein being amphiphilic, forming at least one α-helix, and capable of self-assembling into lipoprotein particles with solubilized lipids when used in the process; or ii) a protein comprising the sequence of SEQ ID NO: 1, 2, 3, 4, 5 or 6 with 1 to 5 amino acids deleted, added, inserted, and / or substituted, said protein being amphiphilic, forming at least one α-helix, and capable of self-assembling into lipoprotein particles with solubilized lipids when used in the process; and The process comprises a) providing a mixture of unpurified membrane vesicles obtained from the cells or organelle membranes of archaea, eukaryotes or prokaryotes, said unpurified membrane vesicles containing both membrane lipids and membrane proteins from the membranes of said unpurified cells or organelles which are the source, b) contacting the mixture of step a) with the lipid-binding polypeptide or derivative thereof in a liquid environment, c) self-assembling the particles.

2. The unpurified membrane vesicles of step a) are a. 1) A step of providing cells and / or cell organelles of the cells; a. 2) A step of lysing or disrupting the cells and / or the cell organelles of the cells; a. 3) A step of obtaining an unpurified membrane fraction; and a. 4) The process according to claim 1, prepared by a step of preparing unpurified membrane vesicles from the unpurified membrane fraction obtained in step a. 3).

3. The process further includes, between step a) and step b), b. 1) Contacting the unpurified membrane vesicles and a surfactant in a liquid environment to obtain a mixture of the unpurified membrane vesicles and the surfactant, and then, in step b), contacting the mixture obtained from step b. 1) with the lipid-binding polypeptide or its derivative in step b), and / or step b) is performed in the presence of a surfactant. The process according to claim 1 or 2.

4. The process according to claim 3, wherein the surfactant is selected from the group consisting of alkylbenzene sulfonate, bile acid, cationic surfactant, nonionic surfactant, zwitterionic surfactant, and mixtures thereof.

5. i) The particles are in a disk shape, ii) The particles have a maximum diameter of 2 nm to 200 nm; iii) The self-assembly of the particles in step c) is performed at a pH of 2.0 to 10.0; and / or iv) The process includes purifying the particles by at least partially removing free membrane lipids, free membrane proteins, free lipid-binding polypeptides, insoluble or aggregated substances, and / or surfactants in step c) or as a subsequent step d). The process according to any one of claims 1 to 4.

6. The process according to any one of claims 1 to 5, wherein the lipid-binding polypeptide is saposin A, saposin B, saposin C, or saposin D, or the derivative is a derivative of saposin A, saposin B, saposin C, or saposin D or a form with a truncated tip.

7. The process according to any one of claims 1 to 6, wherein the particles consist of the at least one lipid-binding polypeptide or its derivative and components of the membrane of the cells or cell organelles derived from the membrane of the cells or cell organelles cited in step a).

8. i) The particles contain membrane lipids derived from the membrane of the cells or the cell organelles cited in step a), and / or ii) The process according to any one of claims 1 to 7, wherein at least a part of said particles contains a membrane protein derived from the membrane of said cells or said organelles cited in step a).

9. The process according to any one of claims 1 to 8, wherein no further lipid other than the components of said unpurified membrane vesicles is added to said process.

10. A process for preparing purified saposin lipo-protein particles, comprising the step of preparing a library according to the process according to any one of claims 1 to 9, f) A further step of purifying at least one type of saposin lipo-protein particle from said library.

11. A library of saposin lipo-protein particles obtainable by the process according to any one of claims 1 to 9, wherein said particles differ in terms of their lipid and / or protein composition.

12. Saposin lipo-protein particles obtainable by the process according to claim 10.

13. The library of particles according to claim 11 or the particles according to claim 12 for use in a diagnostic method, for use in cosmetic treatment or for use as a vaccine preparation.

14. Use of the library of particles according to claim 11 or the particles according to claim 12 as a tool for drug development, drug screening, drug discovery, antibody development, development of therapeutic biopharmaceuticals, for membrane or membrane protein purification, for membrane protein expression, for membrane and / or membrane protein research, for isolation, identification and / or testing of membranes and / or membrane proteins, or for creating a lipidome or proteome database.

Images