PRODUCTION OF SALIPRO PARTICLES

Abstract

The invention relates to a process for preparing saposin lipoprotein particles, comprising a saposin-like protein, lipids and optionally a hydrophobic agent wherein the saposin-like protein or the hydrophobic agent is selectively bound to a support to allow the self-assembly of the saposin lipoprotein particles. The process of the invention comprises the step of a.) providing the hydrophobic agent and lipids, b. 1)/b.2 contacting the hydrophobic agent or the saposin-like protein with a support that is capable of selectively binding either of the two molecules to the support, c.1)/c.2) contacting the support-bound particle components with the remaining particle components, either the saposin-like protein or the hydrophobic agent, to allow for the self-assembly of the saposin lipoprotein particle on the support and d.) optionally eluting the support-bound saposin lipoprotein particles.

Claims

1. Process for producing a saposin lipoprotein particle, wherein the produced saposin lipoprotein particle comprises a saposin-like protein, lipids, and optionally, a hydrophobic agent, wherein the hydrophobic agent is different from the lipids and (I) wherein the process comprises the following steps: a) providing the lipids, and optionally the hydrophobic agent; b.1) contacting the saposin-like protein with a support that is capable of selectively binding the saposin-like protein to the support in a liquid environment; c.1) contacting the support-bound saposin-like protein with the lipids and, optionally, the hydrophobic agent, to allow for the self-assembly of the saposin lipoprotein particle on the support; d) optionally eluting the support-bound saposin lipoprotein particle; or (II) wherein alternatively the process comprises the following steps: a) providing the hydrophobic agent and the lipids; b.2) contacting the hydrophobic agent with a support that is capable of selectively binding the hydrophobic agent to the support; c.2) contacting the support-bound hydrophobic agent with the saposin-like protein to allow for the self-assembly of the saposin lipoprotein particle on the support; d) optionally eluting the support-bound saposin lipoprotein particle.

2. Process according to claim 1, alternative (I), wherein the support comprises a capture moiety, and the saposin-like protein comprises a binding moiety, wherein the capture moiety is capable of selectively binding the binding moiety in the saposin-like protein.

3. Process according to claim 1, alternative (II), wherein the support comprises a capture moiety, and the hydrophobic agent comprises a binding moiety, wherein the capture moiety is capable of selectively binding the binding moiety in the hydrophobic agent.

4. Process according to any one of the preceding claims, wherein the support is in the form of i. beads, ii. a bed, iii. a membrane, and/or iv. a solid support, in particular a solid support with a planar surface.

5. Process according to any one of the preceding claims, wherein the lipids are selected from, the group consisting of viral, archaeal, eukaryotic and prokaryotic lipids, and mixtures thereof.

6. Process according to any one of the preceding claims, wherein in step a) the hydrophobic agent and the lipids are provided in form of a viral, archaeal, eukaryotic or prokaryotic membrane, which comprises the hydrophobic agent and the lipids that are to be incorporated into the saposin lipoprotein particles.

7. Process according to claim 6 in so far as it relates to claim 1, alternative (I), wherein in step c.1) the support-bound saposin-like protein is contacted with the viral, archaeal, eukaryotic or prokaryotic membrane provided in step a) to allow formation of a library of saposin like particles wherein the library comprises a heterogenic mixture of saposin lipoprotein particles with different membrane lipid and optionally membrane protein compositions.

8. Process according to any one of the preceding claims, wherein the saposin-like protein is saposin A, saposin B, saposin C, saposin D or a derivative or truncated form thereof, which is capable of forming saposin lipoprotein particles in the process of claim 1.

9. Process according to claim 8, wherein the derivative or truncated form is selected from i. a protein having at least 20% sequence identity to the full length sequence of SEQ ID NO. 1, 2, 3, 4, 5 or 6; in particular wherein said protein is amphipathic, forms at least one alpha helix, and is capable of self-assembling together with lipids into lipoprotein particles when employed in the process of claim 1; and ii. a protein comprising the sequence of SEQ ID NO. 1, 2, 3, 4, 5 or 6 in which 1 to 40 amino acids have been deleted, added, inserted and/or substituted.

10. Process according to any one of the preceding claims, wherein the hydrophobic agent is selected from the group consisting of a hydrophobic organic compound and a hydrophobic biomolecule.

11. Process according to claim 10, wherein the hydrophobic organic compound and/or the hydrophobic biomolecule is selected from the group consisting of a biologically active agent, a drug, an active ingredient of a drug, an active ingredient of a cosmetic product, an active ingredient of a plant protective product, a dietary and/or nutritional supplement, a diagnostic probe, a contrast agent, a label and an indicator.

12. Process according to any one of claim 10 or 11, wherein the hydrophobic biomolecule is a protein comprising a hydrophobic moiety, in particular a protein selected from the group consisting of a membrane protein, an integral transmembrane protein, an integral monotopic membrane protein, a peripheral membrane protein, an amphitropic protein in a lipid-bound state, a lipid-anchored protein and a chimeric protein with a fused hydrophobic and/or transmembrane domain.

13. Process according to any one of the preceding claims, wherein the hydrophobic agent, the lipids and/or the saposin-like protein is in a detergent-solubilized state and wherein optionally the detergent is selected from the group consisting of alkylbenzenesulfonates or bile acids, cationic detergents and non-ionic or zwitterionic detergents such as lauryl-dimethyl amine-oxides (LDAO), Fos-Cholines, CHAPS/CHAPSO, saponins such as Digitonin and structurally related synthetic detergents such as glycol-diosgenin, alkyl glycosides such as short, medium or longer chain alkyl maltosides, in particular n-Dodecyl β-D-maltoside, glucosides, maltose-neopentyl glycol (MNG) amphiphiles, amphiphilic polymers (amphipols), styrene maleic acid co-polymer (SMA), macrocycle or cyclic oligomers based on a hydroxyalkylation product of a phenol and an aldehyde (Calixarene), and mixtures thereof.

14. Process according to any one of the preceding claims, wherein i. the particles obtained in step c.2) and/or c.1) are disc-shaped, in particular wherein they are disc-shaped and do not comprise a hydrophilic or aqueous core; ii. the particles of step c.2) and/or c.1) have an average maximum diameter of from 2 nm to 200 nm, in particular from 3 nm to 150, preferably from 3 nm to 100 nm; iii. the self-assembly of the particle in step c.2) and/or c.1) is carried out at a pH from 2.0 to 10.0, in particular 6.0 to 10.0, preferably from 6.0 to 9.0, particularly preferred from 7.0 to 9.0, and most preferred from 7.0 to 8.0.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0273] The invention will hereinafter be described with reference to the Figures, which depict certain embodiments of the invention. The invention, however, is as defined in the claims and generally described herein. It should not be limited to the embodiments shown for illustrative purposes in the Figures below.

[0274] FIG. 1 depicts a prior art lipoprotein particle. FIG. 1 is a schematic illustration of the shape and molecular organization of the Apolipoprotein A-1 containing nanosdisc particle (10) of the prior art (EP 1 596 828 B1 discussed above) comprising lipids (3) and apolipoprotein scaffold protein (11).

[0275] FIGS. 2a to 2c are schematic illustrations of Salipro particles in side view (left) and in top view (right) obtained by particular embodiments. In FIGS. 2a, 2b and 2c a Salipro particle comprising a saposin-like protein (2), lipids (3), a membrane protein (4a)/oligomeric membrane protein (4b) and optionally a hydrophobic compound (4c) is shown, wherein the membrane protein (4a,4b) comprises a binding moiety (5).

[0276] FIGS. 3a to 3c are schematic illustrations of Salipro particles in side view (left) and in top view (right) obtained by certain embodiments. In FIGS. 3a, 3b and 3c a Salipro particle comprising saposin-like protein (2), lipids (3) and optionally a membrane protein (4a)/oligomeric membrane protein (4b) is shown, wherein the saposin-like protein (2) comprises a binding moiety (5).

[0277] FIGS. 4a to 4b are schematic illustrations of Salipro particles in side view (left) and in top view (right) obtained by particular embodiments. In FIGS. 4a and 4b a Salipro particle comprising saposin-like protein (2), lipids (3), a hydrophobic compound (4c) and optionally a membrane protein (4a) is shown, wherein the hydrophobic compound (4c) comprises a binding moiety (5).

[0278] FIGS. 5a to 5b are schematic illustrations of Salipro particles in side view (left) and in top view (right) obtained by certain embodiments. In FIGS. 5a and 5b a Salipro particle comprising saposin-like protein (2), lipids (3), a hydrophobic compound (4c) and optionally a membrane protein (4a) is shown, wherein the saposin-like protein (2) comprises a binding moiety (5).

[0279] FIGS. 6a to 6f are schematic illustrations of modes to provide the hydrophobic agent for certain embodiments. In FIGS. 6a and 6d crude membrane vesicles (7, 7′) are depicted, comprising lipids (3) and optionally membrane proteins (4a, 4b), wherein in FIG. 6a the membrane protein (4a) comprises a binding moiety (5). In FIG. 6b and FIG. 6e a membrane protein (4a) associated with lipids (3) and detergent molecules (6) is shown, wherein in FIG. 6b the membrane protein (4a) comprises a binding moiety (5). In FIG. 6c and FIG. 6f a hydrophobic compound (4c) associated with lipids (3) and detergent molecules (6) is depicted, wherein in FIG. 6c the hydrophobic compound (4c) comprises a binding moiety (5).

[0280] FIGS. 7a to 7b are schematic illustrations of modes to provide the saposin-like protein for particular embodiments. In FIGS. 7a and 7b a saposin-like protein (2) is depicted, wherein in FIG. 7a the saposin-like protein (2) comprises a binding moiety (5).

[0281] FIGS. 8a to 8d are schematic illustrations of particular embodiments of hydrophobic agent or saposin-like protein bound to the support in step b.2) and b.1), respectively. In FIG. 8a a saposin-like protein (2) is depicted, which is bound via its binding moiety (5) to the capture moiety (13) of the support (12). In FIG. 8b a hydrophobic compound (4c) associated with lipids (3) and detergent molecules (6) is depicted, which is bound via its binding moiety (5) to the capture moiety (13) of the support (12). In FIG. 8c a membrane protein (4a) associated with lipids (3) and detergent molecules (6).is shown, which is bound via its binding moiety (5) to the capture moiety (13) of the support (12). In FIG. 8d a crude membrane vesicle (7) comprising lipids (3) and membrane proteins (4a, 4b) is shown, which is bound via the binding moiety (5) in the membrane protein (4a) to the capture moiety (13) of the support (12).

[0282] FIGS. 9a to 9c are schematic illustrations of steps b.2) and c.2) of particular embodiments, wherein the hydrophobic agent is bound to the support in step b.2) and contacted with saposin-like protein in step c.2). In FIG. 9a a support(12)-bound membrane protein (4a) associated with lipids (3) and detergent molecules (6) is contacted with saposin-like protein (2) to allow in step c.2) for the self-assembly of a Salipro particle (compare FIG. 2a). In FIG. 9b a support(12)-bound hydrophobic compound (4c) associated with lipids (3) and detergent molecules (6) is contacted with saposin-like protein (2) to allow in step c.2) for the self-assembly of a Salipro particle (compare FIG. 4a). In FIG. 9c a support(12)-bound crude membrane vesicle (7) comprising lipids (3) and membrane proteins (4a, 4b) is contacted with saposin-like protein (2) to allow in step c.2) for the self-assembly of a Salipro particle (compare FIG. 2a).

[0283] FIGS. 10a to 10d are schematic illustrations of steps b.1) and c.1) of particular embodiments, wherein the saposin-like protein is bound to the support in step b.1) and contacted with the hydrophobic agent in step c.1). In FIG. 10a a support-bound (12) saposin-like protein (2) is contacted with a membrane protein (4a) associated with lipids (3) and detergent molecules (6) to allow in step c.1) for the self-assembly of a Salipro particle (compare FIG. 3b). In FIG. 10b a support-bound (12) saposin-like protein (2) is contacted with a crude membrane vesicles (7, 7′) comprising lipids (3) and membrane proteins (4a, 4b) to allow in step c.1) for the self-assembly of Salipro particles and formation of a library of particles (compare FIG. 3a, 3b, 3c). In FIG. 10c a support-bound (12) saposin-like protein (2) is contacted with a hydrophobic compound (4c) associated with lipids (3) and detergent molecules (6) to allow in step c.1) for the self-assembly of a Salipro particle (compare FIG. 5a). In FIG. 10d a support-bound (12) saposin-like protein (2) is contacted with a hydrophobic compound (4c) and membrane protein (4a) both of which are associated with lipids (3) and detergent molecules (6) to allow in step c.1) for the self-assembly of a Salipro particle (compare FIG. 5b).

[0284] FIG. 11 shows the results of Experiment 1 c. FIG. 11 indicates Size Exclusion Chromatography analysis of Salipro particle assembly upon binding of tagged SLC transporter (a membrane protein) to an affinity support and contacting the support-bound SLC transporter with different concentrations of Saposin A.

[0285] FIGS. 12a to 12b shows the results of Experiment 1 d. FIG. 12a indicates Size Exclusion Chromatography analysis of Salipro particles obtained from Sample 5 in Experiment 1 c. The fractions obtained during Size Exclusion Chromatography were analyzed by SDS-PAGE indicating that fractions 13, 14 and 15 mainly contain the assembled Salipro particles. FIG. 12b indicates Size Exclusion Chromatography analysis of fraction 14 as shown in FIG. 12a.

[0286] FIG. 13 shows the results of Experiment 3 c. FIG. 13 indicates Size Exclusion Chromatography analysis of Salipro particle assembly upon binding of the Saposin A to an affinity support and contacting the support-bound Saposin A with additional untagged Saposin A, brain lipids and optionally a hydrophobic agent in form of bacterial ion channel membrane protein T2.

[0287] FIGS. 14a to 14b reproduces FIGS. 4 A and 4 B of Bruhn (2005), Biochem J 389 (15): 249-257.

[0288] The sequences are provided as SEQ ID Nos 7-46 as indicated in table 1 above.

[0289] FIG. 1 depicts a prior art Apolipoprotein A-1 containing nanosdisc particle (10) (see, EP 1 596 828 B1 discussed above) comprising lipids (3) and Apolipoprotein A-1 as lipid binding polypeptide (11). Contrary to the apolipoprotein-derived nanodiscs of the prior art, the lipid binding polypeptide of the present invention, i.e. the saposin-like protein, does not enclose the lipids in a double belt-like fashion (cf. FIGS. 2, 3 and 4) but rather the particles of the invention are held together by a core comprising the membrane lipids which is surrounded by two or more approximately V-shaped or boomerang-shaped lipid binding polypeptides arranged in a head-to-tail orientation with substantially no direct protein-protein contacts between the individual saposin-like proteins within a given Salipro particle obtained by the process of the invention (cf. FIGS. 2, 3 and 4).

[0290] FIG. 2a to 2c are schematic illustrations of Salipro particles obtained according to certain embodiments. The particles of FIGS. 2a, 2b and 2c comprise a saposin-like protein (2), a plurality of different lipids (3), a membrane protein (4a)/oligomeric membrane protein (4b) and optionally a hydrophobic compound (4c). The membrane protein (4a, 4b) in FIGS. 2a to 2c comprises a binding moiety. The binding moiety in the membrane protein can be a natural or engineered binding moiety. The binding moiety of the membrane protein can reside at the N-terminus, C-terminus or within the amino acid sequence of the membrane protein. In case the membrane protein contains an engineered binding moiety, it can be attached to the membrane protein during or after protein synthesis. In another embodiment, which is not depicted, the membrane protein (4a, 4b) can possess multiple binding moieties of the same or different type. The lipids (3) of the Salipro particles depicted in FIG. 2a to 2c differ from each other, meaning that the lipid composition of a Salipro particle is not uniform or homogeneous. Depending on how the membrane protein (4a, 4b) in step a) is provided, the composition of the lipids will vary. In a further embodiment, which is not shown, the Salipro particles may also comprise further components that are typically present in a viral, archaeal, eukaryotic and/or prokaryotic membrane. The lipids (3) and the membrane proteins (4a, 4b) can stem from the same viral, archaeal, eukaryotic or prokaryotic membrane source or from different sources.

[0291] The particles of FIG. 2a to 2c are not drawn to scale. Depending on the size of the membrane protein (4a, 4b) incorporated into the particles, the particles can be substantially different in size compared to other particles. The Salipro particles obtained by the process of the invention are flexible in size. For example, the particle in FIG. 2b harboring an oligomeric membrane protein is larger than and contains more Saposin subunits (2) as compared to the particle in FIG. 2a, which contains a monomeric membrane protein. Depending on the size of the Salipro particles the particles comprise two or more saposin-like molecules per particle, which are arranged in a head-to-tail fashion. The particles depicted in FIGS. 2a and 2c comprise two saposin-like molecules, whereas the particle depicted in FIG. 2b comprises 3 saposin-like molecules.

[0292] FIGS. 2a, 2b and 2c depict—in simplified form as side view and top view—a Salipro particle comprising a saposin-like protein (2), lipids (3) from a viral, archaeal, eukaryotic and/or prokaryotic membrane source, a membrane protein (4a, 4b) and optionally a hydrophobic compound. The membrane protein (4a) can be an integral transmembrane protein in monomeric form. However, the membrane protein can also be an integral transmembrane protein in oligomeric form as depicted in FIG. 2b or a peripheral membrane protein, an amphitropic protein in a lipid-bound state, a lipid-anchored protein or a chimeric protein with a fused hydrophobic and/or transmembrane domain, all of which may be in a monomeric or oligomeric state.

[0293] The particle depicted in FIG. 2c differs from 2a and 2b in that it additionally comprises a hydrophobic compound (4c) of natural or synthetic origin. The number of hydrophobic compounds within one Salipro particle can vary. The hydrophobic compound can form close interactions with the lipids (3) and/or any kind of membrane protein. The membrane protein can for example be a monomeric transmembrane protein (4a) as depicted in FIG. 2c.

[0294] FIG. 3a to 3c depict - again in simplified schematic form (side view left and top view right) and not drawn to scale - Salipro particles obtained according to particular embodiments. The particles depicted in FIGS. 3a, 3b and 3c comprise saposin-like protein (2), lipids (3) and optionally a transmembrane protein of monomeric or oligomeric form (4a, 4b), wherein the saposin-like protein (2) has a binding moiety (5). In another embodiment, which is not shown, the saposin-like protein (2) can possess multiple binding moieties of the same or different type. As described for the particles of FIG. 2 the particles depicted in FIG. 3 can vary regarding their lipid (3) composition and their size to incorporate any kind of hydrophobic protein by simply incorporating more than two saposin-like proteins (2) to form the particle.

[0295] FIGS. 4a and 4b show unscaled schematic illustrations of Salipro particles in side view (left) and top view (right) of certain embodiments of the invention. The particles depicted in FIGS. 4a and 4b comprise a saposin-like protein (2), lipids (3), a hydrophobic compound (4c) and optionally a membrane protein (4a). The hydrophobic compound reveals a binding moiety, which can be of natural or engineered origin. While the natural binding moiety can form an internal part of the hydrophobic compound (4c), the engineered binding moiety is usually attached after synthesis of the hydrophobic compound or after purification of a natural hydrophobic compound to a suitable terminal reactive group. In a further embodiment, which is not shown, the Salipro particles may comprise different kinds of hydrophobic compounds having the same natural or engineered binding moiety. The particles of FIGS. 4a and 4b can vary in the number and kind of lipids (3), hydrophobic compounds (4c), membrane proteins (4a) and saposin-like proteins (2) incorporated into the particles.

[0296] FIGS. 5a and 5b show - again in unscaled and schematic form (side view left and top view right) - Salipro particles of particular embodiments. The particles depicted in FIGS. 5a and 5b differ from the particles depicted in FIGS. 4a and 4b in that the saposin-like protein (2) and not the hydrophobic compound (4c) bears a binding moiety. Regarding different compositions of the particles depicted in FIGS. 5a and 5b, which are not shown, the same considerations apply as described for FIGS. 4a and 4b.

[0297] FIG. 6a to 6f depict particular embodiments which provide the hydrophobic agent in form of a hydrophobic biomolecule (i.e. for example a transmembrane protein in monomeric (4a) or oligomeric (4b) form) or hydrophobic compound in step a) of the process of the invention. In FIG. 6a the membrane proteins (4a, 4b) are provided as crude membrane vesicles (7, 7′). The membrane vesicles (7,7′) can be of viral, archaeal, eukaryotic or prokaryotic origin. They comprise a plurality of lipids (3) and in case of vesicle (7) a plurality of membrane proteins, here exemplified in simplified form by only two membrane proteins (4a, 4b), wherein membrane protein (4a) has a binding moiety (5). Vesicle (7′) is an “empty” lipid-only particle. The crude membrane vesicles, exemplified as vesicles (7, 7′) can be directly obtained by lysing for example a cell or cell organelle of archaeal, prokaryotic or eukaryotic origin. Crude membrane vesicles can also be obtained by rupturing a viral envelope. Vesicles such as (7) and (7′) usually form spontaneously upon lysis or membrane rupture. The crude membrane vesicle can comprise or be associated with detergent molecules (not depicted herein). In FIG. 6b a membrane protein (4a) with a binding moiety (5) is depicted, which is not present within a natural membrane or a crude membrane vesicle, but is in an artificial, detergent-solubilized state (see association of the membrane protein (4a) with lipids (3) and detergent molecules (6) depicted in FIG. 6b). In FIG. 6c a hydrophobic compound with a binding moiety (5) in detergent-solubilized state is depicted, meaning that the hydrophobic compound is embedded within a micelle containing lipids (3) and detergent molecules (6). Optionally, the membrane protein (4a) depicted in FIG. 6b or the hydrophobic compound depicted in FIG. 6c is solubilized by detergent molecules only. The modes to deliver the hydrophobic agents depicted in FIGS. 6d to 6f differ from FIGS. 6a to 6c in that the hydrophobic biomolecule, exemplified as transmembrane protein (4a, 4b) or hydrophobic compound (4c), does not comprise a binding moiety.

[0298] FIGS. 7a and 7b depict particular embodiments to provide the saposin-like protein in the process of the invention. FIG. 7a differs from FIG. 7b in that the saposin-like protein has a binding moiety of natural or engineered origin. While the natural binding moiety forms part of the native amino acid sequence of the saposin-like protein, the binding moiety of engineered origin can be attached during or after synthesis of the saposin-like protein. Engineered binding moieties are optimized to bind with high affinity to their corresponding binding partner, which is the capture moiety in the process of the invention. In a further embodiment, the saposin-like protein can be in association with detergent molecules (not depicted herein).

[0299] FIG. 8a to 8d show unscaled illustrations of support-bound saposin-like protein (FIG. 8a) and hydrophobic agent (FIG. 8b to 8d). The selective binding of the binding moiety in the saposin-like protein (see FIG. 8a) or in the hydrophobic agent (see FIG. 8b to 8d) to the capture moiety (13) of the support (12) is exemplified for a single capture moiety on the support. In reality the support is usually equipped with a plurality of capture moieties of the same or even different type. The interaction of the binding moiety/capture moiety recognition pair can be based on chemical bond formation, affinity based-interactions (including mediation by a bridging agent), hydrophobic interactions and/or electrostatic interactions. The support-bound saposin-like protein (2) can be in a detergent-solubilized state (not depicted herein). Also the crude membrane vesicle (7) can comprise or be associated with detergent molecules (not depicted herein). While the membrane proteins (4a, 4b) of FIG. 8d are embedded in their natural lipid environment in form of a crude membrane vesicle (7), the hydrophobic compound (4c) and the membrane protein (4a) are in an artificial detergent-solubilized state, whereby the micelle around said hydrophobic agents can be formed by lipids and detergents. In a further embodiment, the detergents used to solubilize the saposin-like protein (2) and/or the hydrophobic agents can be of the same or different chemical nature.

[0300] FIG. 9a to 9c show in simplified and schematic form particular embodiments of the process steps b.2) and c.2). The support-bound hydrophobic agent in form of a detergent-solubilized membrane protein (4a) (FIG. 9a), a detergent-solubilized hydrophobic compound (4c) (FIG. 9b) and a membrane protein (4c) embedded in a crude membrane vesicle (7) (FIG. 9c) are contacted with saposin-like protein (2) in step c.2), which is optionally in a detergent-solubilized state. Usually self-assembly of the particles occurs on the support directly upon contacting the support-bound hydrophobic agent with the saposin-like protein (2). Optionally additional solubilized lipids of viral, archaeal, eukaryotic and/or prokaryotic origin are added to the particle assembly reaction, which is not depicted in FIGS. 9a to 9c. The solubilized-membrane protein (4a) of FIG. 9a and the solubilized-hydrophobic compound (4c) are provided in purified form for selective binding to the support in step b.2). The assembled particle of FIGS. 9a and 9b is substantially composed of lipids (3) that have been associated with the solubilized membrane protein (4a) or hydrophobic compound (4c). Optionally, additional lipids are added to the liquid environment of the support. In FIG. 9c a crude membrane vesicle (7) is bound to the support (12). As depicted in FIG. 9c only the membrane protein (4a) bound by its binding moiety (5) to the capture moiety (13) of the support (12) remains bound to the solid support upon contacting with saposin-like protein (2) to allow self-assembly of the Salipro particle on the support. Other membrane proteins, which form initially part of the support-bound crude membrane vesicle in step b.2) and which do not present a complimentary binding moiety for the support's capture moiety, exemplified as multimeric membrane protein (4b) in crude membrane vesicle (7), are not incorporated into the support-bound Salipro particles. The embodiment depicted in FIG. 9c therefore allows the production of Salipro particles comprising a single type of membrane protein (4c) from a plurality of support-bound crude membrane vesicles in step b.2). The assembled particles are substantially free of detergents. The support-bound particles of FIG. 9a to 9c can be eluted. The elution strategy depends on the type of interaction between the capture moiety (13) of the support (12) and the binding moiety (5) in the hydrophobic agent.

[0301] FIG. 10a to 10d show in simplified and schematic form particular embodiments of the process steps b.1) and c.1). The support-bound saposin-like protein is contacted with a detergent-solubilized membrane protein (exemplified as monomeric transmembrane protein (4a), see FIG. 10a), with crude membrane vesicles (exemplified as vesicles (7,7′), see FIG. 10b), a detergent-solubilized hydrophobic compound (4c) (see FIG. 10c) or with both, a detergent-solubilized hydrophobic compound (4c) and a membrane protein (4a) (see FIG. 10d) to allow self-assembly of the respective Salipro particles on the support in step c.1). Neighboring captured saposin-like proteins or additionally added free saposin-like proteins contribute to form an individual Salipro particle (not shown). For example, additional saposin-like protein is added during or after step c.1) to allow for the self-assembly of the saposin lipoprotein particle (not shown). The assembled particle of FIG. 10a is substantially composed of lipids (3) that have been associated with the solubilized membrane protein (4a). The solubilized protein is usually obtained by a protein purification process and is under these circumstances frequently embedded in a micelle comprising detergent molecules and lipids (3). The lipids associated with the solubilized membrane protein are often “shell lipids” of the membrane from which the membrane protein (4a) was purified. The membrane protein may be purified from its “natural” membrane, but this does not have to be the case. For example, a eukaryotic membrane protein can be overexpressed from a transgene in a prokaryotic cell, such as a bacterium, or a virus from which then the membrane protein is purified. The lipids (3) that remain associated with the detergent-solubilized eukaryotic membrane protein might therefore not form part of the “natural” lipid environment of this purified protein. The shell lipids stick tightly to the hydrophobic surfaces of the membrane protein (4a). Only optionally, further lipids are added to the liquid environment for incorporation into the Salipro particles.

[0302] Contacting crude membrane vesicles (exemplified as vesicles (7,7′) in FIG. 10b) with support-bound saposin-like protein in step c.1) (see FIG. 10b) permits production of a support-bound library of Salipro particles, i.e. the support-bound Salipro particles differ in their size and composition comprising saposin-like protein (2), lipids (3) and/or membrane proteins (4a, 4b). Even “empty” particles not comprising any hydrophobic agent can be produced. In the particles of the library the respective membrane protein (4a, 4b) is embedded in the membrane environment from which it was obtained, i.e. the membrane protein remains embedded in its “natural” lipid environment. Thus, the lipids (3) associated with the membrane proteins (4a, 4b) in the assembled Salipro particles are preferably a carry-over from the membrane protein's native lipid environment which is present in the crude membrane vesicle (7). Viral, archaeal, eukaryotic or prokaryotic membranes can serve as source membranes. The assembled particle of FIG. 10c comprises three hydrophobic compounds per particle.

[0303] Of course, the number of hydrophobic compounds incorporated into a single particle can be tuned by adjusting the molar ratio of hydrophobic compounds to lipids employed in the self-assembly reaction of step c.1). The hydrophobic compound of FIG. 10c is added to the self-assembly reaction in a solubilized state. Solubilization of hydrophobic compounds is usually achieved by detergent molecules, similar to the solubilization of hydrophobic membrane proteins. As depicted in FIG. 10c lipids can form part of the solubilized state of the hydrophobic compound. Optionally additional lipids can be added. As depicted in FIG. 11d the hydrophobic agents in form of the solubilized hydrophobic compound (4c) and the membrane protein (4a) can be applied together in step c.1). Any desired molecular ratio of solubilized hydrophobic compound to membrane protein can be chosen which influences the composition of the obtained particles. The assembled particles depicted in FIGS. 10a to 10d are usually substantially free of detergents. The support-bound particles of FIG. 10a to 10d can be eluted. The elution strategy depends on the type of interaction between the capture moiety (13)/binding moiety (5) pair.

EXAMPLES

[0304] The following example serves to further explain the invention in more detail, specifically with reference to certain embodiments and Figures which, however, are not intended to limit the present disclosure.

I Abbreviations

[0305] The following abbreviations will be used:

[0306] Asp Aspartic acid

[0307] CV column volume

[0308] daGFP This kind of green fluorescent protein can be used in the same way as normal

[0309] GFP using argon laser based or UV based excitation apparatus to allow the detection of fluorescence. The protein has a peak excitation of 510 nm and a peak emission of 521nm.

[0310] EB1 elution buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 400 mM Imidazol)

[0311] EB2 elution buffer (50mM HEPES pH 7.5, 2% DDM, 0.4% CHS, 200mM NaCl, 1mM L-Asp, 1mM EDTA, 1mM TCEP and 5% Glycerol, 2.5mM desthiobiotin)

[0312] EB3 50 mM HEPES pH 7.5, 200 mM NaCl, 10% glycerol, 250 ug/mL FLAG-peptide

[0313] EB4 50 mM HEPES pH 7.5, 200 mM.NaCl, 10% glycerol supplemented with 2mM biotin

[0314] EDTA ethylenediaminetetraacetic acid

[0315] DDM n-dodecyl-β-D-maltopyranoside

[0316] GF gel filtration buffer (20 mM HEPES pH 7.5, 150 mM NaCl)

[0317] HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

[0318] His Histidine

[0319] HNG buffer 50mM HEPES pH 7.5, 200mM NaCl and 5% glycerol

[0320] HNG buffer II 50mM HEPES pH 7.5, 200mM NaCl and 10% glycerol

[0321] IMAC immobilized metal affinity chromatography

[0322] IPTG isopropyl β-D-1-thiogalactopyranoside

[0323] LB1 20 mM HEPES pH 7.5, 150 mM NaCl, 20 mM Imidazol

[0324] LB2 50 mM HEPES/Tris-base, pH 7.4, 50 mM NaCl buffer supplemented with 1 mM L-Asp, 1 mM EDTA, 1 mM PMSF, 1 mM TCEP, and 1:200 (v/v) dilution of mammalian protease inhibitor cocktail (Sigma)

[0325] PEI poly-ethylenimine

[0326] PMSF phenylmethylsulfonyl fluoride

[0327] SB solubilization buffer

[0328] SEC size-exclusion chromatography

[0329] SLC solute carrier

[0330] TCEP tris(2-carboxyethyl)phosphine

[0331] TEV Tobacco etch virus

[0332] Tris tris(hydroxymethyl)aminomethane

[0333] TB medium terrific broth medium

[0334] WB working buffer (50mM HEPES pH 7.5, 2% DDM, 0.4% CHS, 200mM NaCl, 1mM L-Asp, 1mM EDTA, 1mM TCEP and 5% Glycerol)

II Purification of saposin A

[0335] Purified saposin A used in the below experiments was prepared as follows. Saposin A protein expression was carried out using a vector with the coding region for human saposin A (SEQ ID NO: 1) inserted into a pNIC-Bsa4 plasmid and transformed and expressed in E. coli Rosetta gami-2 (DE3) (Novagen) strains. Cells were grown at 37°0 C. in TB medium supplemented with Tetracycline, Chloramphenicol and Kanamycin and induced with 0.7 mM IPTG. Three hours after induction, the cells were collected by centrifugation at 12.000×g for 15 min. The supernatant was discarded, the cell pellet was resuspended using lysis buffer LB1 (20 mM HEPES pH 7.5, 150 mM NaCl, 20 mM Imidazol) and disrupted by sonication. Lysates were subjected to centrifugation at 26.000×g for 30 min, the supernatant heated to 85°0 C. for 10 min, followed by an additional centrifugation step at 26.000×g for 30 min. Preparative IMAC purification was performed by batch-adsorption of the supernatant by end-over-end rotation with Ni Sepharose™ 6 Fast Flow medium for 60 min. After binding of saposin A to the IMAC resin, the chromatography medium was packed in a 10-mm-(i.d.) open gravity flow column and unbound proteins were removed by washing with 15 bed volumes of lysis buffer LB1. The resin was washed with 15 bed volumes of wash buffer (20 mM HEPES pH 7.5, 150 mM NaC1, 40 mM Imidazol). Saposin A was eluted by addition of five bed volumes of elution buffer EB1 (20 mM HEPES pH 7.5, 150 mM NaCl, 400 mM Imidazol). 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 an un-cleavable His-tag was removed from the eluate by passing it over 2 ml IMAC resin. Cleaved target proteins were concentrated to a volume of 5 ml using centrifugal filter units and loaded onto a HiLoad Superdex™ 200 16/60 GL column using an AKTAexplorer™ 10 chromatography system (both GE Healthcare). Peak fractions were pooled and concentrated to 1.2 mg/ml protein. The protein sample was flash frozen in liquid nitrogen and stored at −80 C.

III Generation of Salipro Particles on Support

Example 1

[0336] In this example, a large transmembrane transporter (SLC) is used as hydrophobic agent in alternative (II) of the process according to the invention. The lipids and the hydrophobic agent are provided in the form of a crude membrane fraction obtained from SLC-overexpressing HEK293F cells. The SLC transporter contains a Strep II-tag as binding moiety. Anti-Strep-II affinity purification beads, were used as support according to the invention. They contain anti-Strep-II capture moieties that are capable of binding the Strep II-binding moiety comprised in the SLC transporter protein. Addition of Saposin A to the support-bound SLC transporter-containing solubilized membranes allowed formation of SLC-transporter-containing saposin lipid particles that were still attached to the support via the Strep-II tag comprised in the SLC transporter protein. Thus, the assembly of the saposin lipid particles took place entirely on the support and with the endogenous lipids that were derived from the cellular membrane and still complexed with the support-bound SLC transporter protein.

[0337] 1.a. Over-Expression of Membrane Protein

[0338] The coding sequence of human SLC transporter was introduced into an expression vector encoding for an N-terminal Strep-tag II followed by daGFP and a PreScission protease cleavage site. Prior to transfection, HEK293F cells (ATCC cell line, myco-plasma test negative) were grown in Exce11293 medium (Sigma) supplemented with 4 mM L-glutamine (Sigma) and 5μg m1.sup.-1 Phenol red (Sigma-Aldrich) to densities of 2.5×10.sup.6 cells ml.sup.−1. Cells were transiently transfected with the expression vector in Freestyle293 medium (Invitrogen) using poly-ethylenimine (PEI) (Polysciences) at a density of 2.5×10.sup.6 cells m1.sup.-1, diluted with an equivalent volume of Exce11293 6 h after transfection, and treated with 2.2 mM valproic acid (Sigma) 12 h after dilution of the cultures. Transfected cells then overexpressed the fusion protein Strep II-daGFP-SLC. All cells were collected at around 48 h after transfection.

[0339] 1.b. Preparation of Crude Cell Membranes

[0340] Large-scale expression of the fusion protein Strep II-daGFP-SLC was performed in a 51 culture essentially as described in a. above. Cells were collected in lysis buffer (LB2) containing 50 mM HEPES/Tris-base, pH 7.4, 50 mM NaCl supplemented with 1 mM L-Asp, 1 mM EDTA, 1 mM PMSF, 1 mM TCEP, and 1:200 (v/v) dilution of mammalian protease inhibitor cocktail (Sigma), and disrupted in a cell homogenizer (EmulsiFlex-05, Avestin) via 3 runs at approximately 103,000 kPa. The resulting homogenate was clarified by centrifugation (4,500 g for 0.5 h) and the crude membranes were collected by ultracentrifugation (186,000 g for L5 h). Membranes were washed once with the LB2 buffer and finally homogenized with a douncer in a buffer containing 50 mM HEPES/Tris-base, pH 7.4, 200 mM NaCl, 1 mM L-Asp, 1 mM EDTA, 1 mM TCEP, and 10% glycerol, snap-frozen in liquid N.sub.2 and stored at −80°0 C. at 0.5 g membranes ml.sup.−1.

[0341] 1.c. Binding to Affinity Support and Elution

[0342] The following buffers were used: [0343] Solubilization buffer (SB): 50 mM HEPES pH 7.5, 2% DDM, 0.4% CHS, 200 mM NaCl, 1 mM L-Asp, 1 mM EDTA, 1 mM TCEP and 5% Glycerol. [0344] Working buffer (WB): 50 mM HEPES pH 7.5, 200 mM NaCl, 1 mM L-Asp, 1 mM TCEP and 5% glycerol. [0345] Elution buffer EB2: WB supplemented with 2.5 mM desthiobiotin (dBiotin). [0346] HNG buffer: 50 mM HEPES pH 7.5, 200 mM NaCl and 5% glycerol.

[0347] 800 μl crude membranes (0.5 g membranes m1.sup.-1) containing over-expressed SLC transporter were solubilized with 4.2 ml SB and incubated for 90 min at 4°0 C. using a rotating wheel. Membrane debris was removed by centrifugation at 30000 g for 30 min followed by the addition of 900 μl equilibrated anti-Strep-II affinity purification beads (StrepTactin Sepharose beads, GE healthcare) and the total volume was corrected to 5 ml using WB. The sample was then incubated at 4°0 C. for 1 h to allow binding of the Strep-II tagged SLC transporter to the affinity beads. The sample was then divided onto 5 separate columns allowing to remove non-bound material by gravity flow through. The affinity beads were not washed at this stage and contained the affinity bound SCL transporter in an environment (“dead volume” of the beads) partly containing native cell membrane lipids, detergent micelles and the WB components.

[0348] Different amounts (0-4 ml) of 3.6 mg/ml Saposin were added to the corresponding columns. The mixtures were then transferred to five new tubes and the total sample volumes were corrected to 5 ml using WB as follows: [0349] Sample 1: 0 ml Saposin A +4 ml WB [0350] Sample 2: 0.5 ml Saposin A +3.5 ml WB [0351] Sample 3: 1 ml Saposin A +3 ml WB [0352] Sample 4: 2 ml Saposin A +2 ml WB [0353] Sample 5: 4 ml Saposin A

[0354] Samples 1-5 were then incubated for 1 h at 4°0 C. using a rotating wheel, before being transferred back to columns. Non-bound material was removed from the column using gravity flow through, followed by 6 CV washes in WB and an elution step with 4 ml elution buffer EB2. 50 μI of each eluted sample was analyzed using SEC (protein detection at 280 nm) with a Superose 6 increase, 5/150 GL column running in detergent free WB as SEC buffer.

[0355] The results are depicted in FIG. 11. The results demonstrate that saposin lipoprotein particles can be obtained even if the membrane protein, which is in complex with lipids, is bound to an affinity support. Increasing amounts of Saposin A (0.5 mL, 1 mL, 2 mL and 4 mL of Saposin A in a concentration of 3.6 mg/me also lead to increased formation of Salipro particles. As a negative control under these conditions, no Salipro particles could be obtained when Saposin A was excluded from the liquid environment (FIG. 11, sample 1).

[0356] The results depicted in FIG. 11 also reinforce that prior to elution with detergent-free buffer the support-bound Salipro particles are stable during the thorough washing steps of the column.

[0357] 1.d. Analysis of Obtained Salipro Particles

[0358] The eluate obtained after incubation of the affinity beads with 4 ml SapA (see previous section c, sample 5) was concentrated using Amicon Ultra-2 centrifugal filter with a 100 kDa molecular size cut-off. 40 μl of the concentrated sample was further analyzed by SEC using a Superose 6 increase 5/150 GL column in a detergent-free HNG buffer supplemented with 1 mM L-Asp.

[0359] Analysis of the SEC fractions by SDS-PAGE indicates (FIG. 12a) that mainly fractions 13, 14 and 15 contain purified Salipro particles, containing the SLC transporter and Saposin.

[0360] To further validate the homogeneity of the reconstituted Salipro particles, 20 μI from fraction 14 were further analyzed by SEC using a Superose 6 increase 5/150 GL column in detergent free-HNG buffer supplemented with 1 mM L-Asp. The corresponding SEC profile (FIG. 12b) further demonstrates the stability and homogeneity of the Salipro particles when reconstituted on the affinity beads.

[0361] The data presented herein clearly demonstrate that it is possible to reconstitute hydrophobic agents into Saposin particles while one of the particle components is bound to an affinity support. The data also shows that crude membranes can be used in the process of the invention.

IV Generation of Salipro Particles from Whole Cells

Example 2

[0362] In this example, a membrane protein is used as hydrophobic agent in alternative (II) of the process according to the invention. The lipids and the hydrophobic agent are provided in the form of intact cells, i.e. human embryonic kidney (HEK) cells, overexpressing the membrane protein. Said HEK cells are only contacted with a detergent without performing a mechanical cell lysis step. The eukaryotic membrane protein contains a FLAG-tag as binding moiety. Anti-FLAG affinity purification beads are used as support according to the invention. They contain anti-FLAG capture moieties that are capable of binding the FLAG-binding moiety comprised in the eukaryotic membrane protein. Addition of Saposin A to the support-bound eukaryotic membrane protein comprised in the detergent-treated membranes allows formation of saposin lipid particles containing the eukaryotic membrane protein. Thus, in this example, the assembly of the saposin lipid particles takes place entirely on the support and with the endogenous lipids that are provided in the form of detergent-treated whole cells expressing the to-be-included eukaryotic membrane protein of interest.

[0363] 2.a. Over-Expression of Membrane Protein

[0364] The coding sequence of the eukaryotic membrane protein is introduced into an expression vector encoding for an N-terminal FLAG-tag. Prior to transfection, HEK 293F cells are grown in 293 Freestyle culture media and transfected using the PEI-Max reagent using the protocol provided by the manufacturers (ThermoFisher). Transfected cells then overexpress the membrane protein. All cells are collected at around 48 hours_post transfection.

[0365] 2.b. Preparation of Solubilized Membranes

[0366] The cells overexpressing the FLAG-tagged eukaryotic membrane protein are harvested to a cell pellet. The cell pellet is then dissolved in HNG buffer II, additionally comprising a 25× protein inhibitor cocktail at a final concentration of 2×. Subsequently, a solution comprising 10% GDN in water (w/v) is added to the resuspended cells to a final concentration of 1% GDN (w/v). The sample is then incubated on a rotating wheel in a cold cabinet for 5 min. Afterwards, the sample is centrifuged at 5000 g at 4°0 C. for 5 min. The supernatant comprising the solubilized material, including the detergent-treated membranes, is recovered and incubated for another 50 min on a rotating wheel in a cold cabinet. After this incubation step, the supernatant is centrifuged at 30000 g and 4°0 C. for 30 min to remove membrane debris and then used in the next step 2.c for binding to the affinity support.

[0367] 2.c. Binding to Affinity Support and Elution

[0368] The following buffers are used: [0369] HNG buffer II: 50 mM HEPES pH 7.5, 200 mM NaCl and 10% glycerol [0370] EB3: 50 mM HEPES pH 7.5, 200 mM NaCl, 10% glycerol, 250 μg/mL FLAG-peptide

[0371] 4 columns allowing to remove non-bound material by gravity flow through are prepared by loading each column with 100 μl of equilibrated M2 anti-FLAG affinity purification beads (SigmaAldrich. Afterwards 500 μl of solubilized membranes obtained in step 2.b are added to each column. The flow-through is then re-passed three times through the column to allow efficient binding of the FLAG-tagged eukaryotic membrane protein to the affinity beads. The affinity beads loaded with the FLAG-tagged eukaryoticmembrane protein are not washed at this stage and contain the affinity bound eukaryotic membrane protein in an environment (“dead volume” of the beads) partly containing native cell membrane lipids and detergent micelles and HNG buffer II components.

[0372] Different amounts (0-6 ml) of 1 mg/ml Saposin A are added to the corresponding columns. [0373] Sample 1: 1 ml HNG buffer II [0374] Sample 2: 1 ml Saposin A [0375] Sample 3: 3 ml Saposin A [0376] Sample 4: 6 ml Saposin A

[0377] The mixtures are then transferred to four new tubes and incubated for 25 min at 4°0 C. using a rotating wheel, before being transferred back to the columns. Non-bound material is removed from the column using gravity flow through, followed by 10 CV washes in HNG buffer II and an elution step with 500 μl elution buffer EB3.

[0378] 2.d. Analysis of Salipro Particles

[0379] The eluates obtained after incubation of the affinity beads with different amounts of SapA (see previous section 2.c, samples 1 to 4) are concentrated using Amicon Ultra-2 centrifugal filters (10 kDa NMWL) at 13000 g and 4°0 C. The concentrated samples are further analyzed by SEC using a Superose 6 increase 5/150 GL column in a detergent-free HNG buffer II to detect formed Salipro particles.

[0380] It is expected that with the aforementioned experimental workflow saposin lipoprotein particles can be obtained from intact cells as starting material, which have not been subjected to a mechanical cell lysing and while the eukaryotic membrane protein of interest is bound to an affinity support. As a negative control under these conditions, no Salipro particles should be obtained when Saposin A is excluded from the liquid environment.

V Generation of Salipro Particles on Support

Example 3

[0381] In this example, the reconstitution of Salipro particles was carried out according to alternative (I) of the process of the invention, i.e. Saposin was immobilized on an affinity support. To this end, Saposin was biotinylated and bound to an avidin affinity bead matrix. Contacting the support-bound Saposin with additional untagged Saposin, lipids and optionally a hydrophobic agent allowed formation of Salipro particles according to the invention. Thus, assembly of the saposin lipid particles took place on the support.

[0382] 3a. Preparation of Biotinylated Saposin A

[0383] Saposin A was biotinylated using EZ-Link®NHS-Biotin Reagents (Thermo Fisher, reference 21343) according to the manufacturer's protocol. Quantification of the biotin number per Saposin A was then performed with Quant*Tag Biotin Kit (Vector laboratory, BDK-2000) and showed that 1.1 biotins per Saposin A molecule were present.

[0384] 3.b. Binding to Affinity Support and Elution

[0385] Monomeric avidin matrix (Thermo Fisher 20228) was prepared and washed according to the manufacturer's protocol. The biotinylated Saposin A was bound to the prepared avidin affinity matrix.

[0386] For each sample, 100 μl of biotinylated Saposin A (1.2 mg/ml) were bound to 25 p.I of avidin affinity matrix by passing the biotinylated Saposin A three times over the matrix, which was contained in a column (BioRad, Polyprep Chromatography column, art.nr 7311550). The affinity matrix was then extensively washed with HNG buffer II to ensure removal of non-bound Saposin A.

[0387] With the Saposin A loaded avidin affinity matrix, two different particle assembly conditions were evaluated: In sample 1, brain lipids and untagged Sapsosin A were added to the affinity resin with pre-immobilized Saposin A. In sample 2, brain lipids, a membrane protein (bacterial ion channel membrane protein T2) and untagged Saposin A were added to affinity resin with pre-immobilized Saposin A.

[0388] The brain lipid solution was prepared by dissolving 5 mg/ml brain lipids (Sigma-Aldrich) in 0,5% DDM and pre-incubated 5 minutes at 37° C.

[0389] The bacterial ion channel membrane protein T2 was purified as previously described in F Guettou et al., Nature structural & Molecular Biology, 21; 728-731, 2014.

[0390] The particle assembly conditions for samples 1 to 2 were as follows: [0391] Sample 1: 16 μl brain lipid solution were added to the affinity resin with pre-immobilized Saposin A and incubated 5 min at room temperature before adding 100 μl non-tagged Saposin A (1.2 mg/ml). [0392] Sample 2: 16 μl brain lipid solution were mixed with 8 μl T2 (10 mg/m1) and incubated 5 min at 37°0 C., before adding the mixture to the affinity resin with pre-immobilized Saposin A. The sample was then incubated at room temperature for 5 min before adding 100 μl non-tagged Saposin A (1.2 mg/ml).

[0393] The two samples were then incubated simultaneously at room temperature on a rotating wheel for 25 min. Subsequently, the following buffers were used to treat the sample columns: [0394] HNG buffer II: 50 mM HEPES pH 7.5, 150 mM NaCl and 10% glycerol [0395] EB4: HNG buffer supplemented with 2mM biotin (Thermo Fisher 29129)

[0396] The affinity beads were washed extensively with detergent-free HNG buffer II (3 times using 10 CV) and immobilized samples were eluted using the elution buffer EB4.

[0397] 3.c. Analysis of the obtained Salipro particles

[0398] The eluted samples were subjected to analytic SEC, using a Superdex™ 200 5/150 GL analytical gel filtration column running in HNG buffer II.

[0399] The results are shown in FIG. 13. For sample 1 and 2 Salipro particles were detected in the elution profile (see SEC peak at 6.4 min for sample 1 and SEC peak at 4.5 min for sample 2 of FIG. 13). Thus, the immobilized Saposin A enabled Salipro particle assembly to take place on the affinity support.

[0400] Altogether, the data presented herein clearly demonstrate that it is possible to reconstitute Salipro particles using different starting materials while one of the particle components is bound to an affinity support.