WO2021152584A1

WO2021152584A1 - Methods of analyzing cell membranes

Info

Publication number: WO2021152584A1
Application number: PCT/IL2021/050093
Authority: WO
Inventors: Thien TRUONG VAN; Guy Patchornik; Mordechai Sheves
Original assignee: Ariel Scientific Innovations Ltd.; Yeda Research And Development Co. Ltd.
Priority date: 2020-01-28
Filing date: 2021-01-27
Publication date: 2021-08-05

Abstract

A method of precipitating nanostructures out of a mixture of cellular components is disclosed. The nanostructures have an outer surface comprising phospholipids. The method comprises contacting the mixture with an oligo-amine having a sufficient number of amine groups such that it ionically conjugates with said nanostructures.

Description

METHODS OF ANALYZING CELL MEMBRANES

RELATED APPLICATIONS

This application claims the benefit of priority of US Patent Application No. 62/966,619 filed 28 January, 2020, the contents of which are incorporated herein by reference in their entirety.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to methods for precipitating nanostructures and in some embodiments to the purification of proteins situated therein.

Isolation of membrane proteins (MPs) in a pure, concentrated and functional state is a precondition for structural determination by X-ray crystallography, electron microscopy or nuclear magnetic resonance (NMR).

Extraction of membrane proteins from the membrane of a cell in which they are expressed may be achieved by addition of detergents at concentrations greater than their critical micellar concentration (cmc). Under these conditions, the detergent disrupts the membrane and, in parallel, surrounds and covers the hydrophobic domains of the protein, leading to formation of water- soluble [detergent-MP-lipid] ternary complexes. Purification is accomplished either via classical chromatographic methods ( e.g . ion exchange chromatography) or by genetically engineered affinity-tags (e.g. His-tag) which can lead to highly pure protein preparations. Clearly, exclusion of other (non-membrane) cellular proteins by non-chromatographic means prior to the chromatographic step, would simplify purification and potentially lead to higher recovery yields and overall greater purity.

In 1981, Bordier demonstrated that MPs (being hydrophobic) partition efficiently into detergent-rich phases composed of the non-ionic detergent Triton X-114, whereas water-soluble proteins do not. This partitioning process, called cloud point extraction, relied on the ability of Triton X-l 14 to undergo phase separation at ~22°C into detergent-rich and detergent-poor phases. Although Triton X-114 provided working conditions that could preserve the functionality of many MPs, the approach was limited by the fact that numerous other detergents, commonly used in the purification of membrane proteins, only reach the cloud point at elevated temperatures that would denature most proteins.

Background art includes Patchomik et ah, Journal of Colloid and Interface Science 388 (2012) 300-305; and Patchomik et al., Soft Matter, 2012, 8, 8456.

Additional background art includes W02005/010141, W02006/085321 and

W02009/010976 and US Patent No. 10,030,224. SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present invention there is provided a method of precipitating nanostructures out of a mixture of cellular components, the nanostructures having an outer surface comprising phospholipids, the method comprising contacting the mixture with an oligo-amine having a sufficient number of amine groups such that it ionically conjugates with the nanostructures, thereby precipitating nanostructures out of a mixture of cellular components.

According to an aspect of some embodiments of the present invention there is provided a method of purifying a membrane protein comprising: (a) precipitating cell membrane fragments which comprise the membrane protein from a cell lysate according to the method described herein;

(b) isolating the cell membrane fragments following the precipitating; and

(c) solubilizing the membrane protein, thereby purifying the membrane protein.

According to an aspect of some embodiments of the present invention there is provided a method of precipitating microstructures having an outer surface comprising phospholipids, the method comprising contacting the microstructures with an oligo-amine having a sufficient number of amine groups such that it ionically conjugates with the nanostructures, thereby precipitating microstructures.

According to some embodiments of the invention, the oligo-amine comprises between 3 and 20 amine groups.

According to some embodiments of the invention, the oligo-amine comprises between 3 and 6 amine groups.

According to some embodiments of the invention, the outer surface of the nanostructures comprise a protein. According to some embodiments of the invention, the nanostructures comprise cell membrane fragments and the mixture of cellular components is a cell lysate.

According to some embodiments of the invention, the membrane fragments comprise a membrane protein.

According to some embodiments of the invention, the nanostructures comprise liposomes. According to some embodiments of the invention, the nanostructures are synthetic nanostructures.

According to some embodiments of the invention, the nanostructures comprise exosomes.

According to some embodiments of the invention, the oligo-amine has a molecular weight below 2,000 Daltons. According to some embodiments of the invention, the oligo-amine is a non-aromatic oligo- amine.

According to some embodiments of the invention, the oligo-amine is a linear oligo-amine.

According to some embodiments of the invention, the linear oligo-amine is selected from the group consisting of pentaethylenehexamine, tetraethylenepentamine pentahydrochloride and triethylenetetramine hydrate.

According to some embodiments of the invention, the oligo-amine comprises a quaternary ammonium.

According to some embodiments of the invention, the oligo-amine is a ring-shaped oligo- amine.

According to some embodiments of the invention, the cell membrane fragments are generated by sonicating whole cells.

According to some embodiments of the invention, the cell lysate is a whole cell lysate.

According to some embodiments of the invention, the cell lysate is devoid of organelles greater than about 2 microns.

According to some embodiments of the invention, the method further comprises isolating the nanostructures from the mixture following the precipitating.

According to some embodiments of the invention, the method further comprises solubilizing proteins of the cell membrane fragments following the isolating.

According to some embodiments of the invention, the solubilizing is effected with a detergent.

According to some embodiments of the invention, the detergent comprises a non-ionic detergent.

According to some embodiments of the invention, the detergent is selected from the group consisting of decyl //-D-maltoside (DM), dodecyl //-D-maltoside (DDM), octyl //-D-glucoside (OG) and octyl //-D- l -thioglucoside (OTG).

According to some embodiments of the invention, the contacting is effected in the absence of a detergent.

According to some embodiments of the invention, the contacting is effected in the absence of a hydrophobic chelator.

According to some embodiments of the invention, the cell membrane fragments have a length ranging between 200-700nm.

According to some embodiments of the invention, the cell lysate is derived from a bacterial cell. According to some embodiments of the invention, the cell lysate is derived from an archaeal cell.

According to some embodiments of the invention, the archaeal cell is a halobacterium cell.

According to some embodiments of the invention, the halobacterium is selected from the group consisting of Halobacterium salinarum, Haloferax denitrificans, Alorubrum distributum, Alobacterium salinarum, Halobacterium jilantaiense, Halorubrum lacusprofundi, Haloferax mediterranei, Halobacterium noricense, Natronomonas pharaonis, Halobacterium piscisalsi, Halorubrum saccharovoru, Halobacterium salinarum, Halorubrum sodomense, Halorubrum trapanicum, Haloarcula vallismortis and Halobacterium volcanii. According to some embodiments of the invention, the solubilizing is effected using a detergent.

According to some embodiments of the invention, the detergent is a non-ionic detergent.

According to some embodiments of the invention, the isolating comprises:

(a) centrifuging the cell lysate to form a pellet comprising the cell membrane fragments and a supernatant; and

(b) removing the supernatant from the pellet. According to some embodiments of the invention, the membrane protein is a retinyledene protein.

According to some embodiments of the invention, the retinyledene protein is selected from the group consisting of channelrhodopsin, bacteriorhodopsin, halorhodopsin, and proteorhodopsin. According to some embodiments of the invention, the oligo-amine comprises between 3 and 20 amine groups.

According to some embodiments of the invention, the microstructures comprise biological cells.

Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

FIG. 1. Schematic illustration of membrane clustering in the presence of positively charged oligo-amines. Phospholipid bilayers containing embedded membrane proteins (MPs) are reversibly clustered upon addition of linear, positively charged oligo-amines capable of forming electrostatic interactions with negatively charged side chains of the MP (e.g. aspartic acid, glutamic acid) and /or with phospholipid head groups carrying a net negative charge.

FIGs. 2A-B Spectroscopic analysis of the active center and secondary structure of the purple membrane protein, bacteriorhodopsin (bR), in the presence of oligo-amines. A. Oligo- amines (4 mM) with indicated charge were added to a suspension (pH 7, 50 mM Tris-HCl) of purple membranes as described in the Experimental section. UV-Vis spectra were measured after 1 hour incubation at 19°C in the dark. B: CD spectra of a suspension (pH 7, 50 mM Tris-HCl) of purple membranes in the presence of oligo-amines at zero time and after 2 hours at 19 °C in the dark. For both parts A and B, the control, i.e. in the absence of oligo-amines, is labelled [C]

FIGs. 3A-B. Light microscope images of purple membranes at indicated time points in the presence or absence of oligo-amines exhibiting 3, 4, 5 or 6 positive charges. Scale bar (250 pm) applies to all images. B. Centrifuged (5 min. at 326 x g) Eppendorf™ tubes containing purple membranes without (left) and shortly after the addition of (right) the +6 oligo-amine analog (12.5 mM) at pH 7 (20 mM NaPi).

FIGs. 4A-B. Light microscopy images of purple membrane suspensions (pH 7, 20 mM NaPi) conjugated first with indicated for 15 minutes in the dark and at 19 °C and followed by the addition of NaCl. Photographs were taken either 15 minutes or 3 hours after addition of NaCl. Scale bar (250 pm) applies to all images. Whereas 0.5 M of NaCl and 3 hours of incubation were required to dissociate membrane aggregates conjugated via an oligo-amine with +6 charges (Figure 4A), only 50-100 mM of NaCl and 15 minutes were needed to dissolve membrane aggregates conjugated via an oligo-amine with +5 charges (Figure 4B). This finding is in agreement with the greater binding affinity of the +6 oligo-amine to surface exposed negative charges on the membrane (or protein) relative to the +5 oligo-amine analog.

FIG. 5. Effect of reduced pH on purple membrane conjugation via oligo-amines. Purple membranes were conjugated with 2 mM oligo-amines at pH 7 (50 mM Tris) and at pH 4 and 2.5 in the presence of 50 mM AcOH and 50 mM Glycine, respectively.

FIGs. 6A-E. Scanning electron microscope (SEM) images of purple membranes deposited from aqueous suspension (pH 7, 50 mM Tris-HCl) without (A) - and with (B-E) oligo-amines (+3 to +6) - as described in the Experimental section.

FIGs. 7A-B. Conjugation of bR-vesicles with +6 oligo-amines. SEM images of vesicles comprising bR protein, native phospholipids and the non-ionic detergent OTG in the presence of the +6 oligo-amine (10 mM) and 5%w/v PEG-6000, pH 7. Arrows indicate possible fusion between 2 or 3 vesicles. In the absence of the oligo-amine, no vesicle dimers or trimers were observed. Figure 7B represents a higher magnification of Figure 7A.

FIGs. 8A-B. Conjugation of xanthrorhodopsin (XR) protein-containing membranes via linear oligo-amine (+6) molecules. A. UV-Vis absorption spectra of suspensions (20 mM NaPi, pH 7) of XR-membranes without [-] or with [+] 12.5 mM (+6) oligo-amine. Measurements were made following lhr incubation at 19 °C in the dark. B. Images of centrifuge pellets (5 minutes at 326 x g) in Eppendorf™ tubes containing suspensions (20 mM NaPi, pH 7) of XR-membranes without control or with 12.5 mM (+6) oligo-amine. Measurements were made following 1 hour incubation at 19 °C in the dark.

FIGs. 9A-F. Scanning electron microscope (SEM) images of XR-protein containing native membranes deposited from aqueous suspension (pH 7, 50 mM Tris-HCL) without (A) - and with (B-F) oligo-amines (+3 to +6) - as described in the Experimental section.

FIG. 10. SDS-PAGE analysis. Lane 1: E. coli lysate serving as an artificial contamination background; Lane 2: Pure purple membranes containing the target membrane protein: bacteriorhodopsin (bR); Lane 3 : Mixture of lysate and bR used as the starting material for process demonstration; Lanes 4-5: Supernatant and pellet composition, respectively, after a short incubation (1 minute) and spin (376xg-, 5 minutes) in the absence of the oligo-amine: PEHA as described above; Lanes 6-7, as in lanes 4-5, but in the presence of 4 mM PEHA. The chemical composition of the oligo-amine is shown on the right. The gel is Coomassie stained.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to methods for precipitating nanostructures and in some embodiments to the purification of proteins situated therein. Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

Purification of membrane proteins requires disruption of the cell membrane. Typically, this is carried out using detergents. However, such agents may be deleterious to the proteins, causing denaturation.

The present inventors have now conceived of a method of membrane conjugation that does not require the use of detergents (during removal of most water-soluble proteins from the system), requires no sophisticated instrumentation (e.g. ultracentrifugators), is relatively rapid (e.g. few minutes), is performed under mild conditions and is capable of removing the majority of non membrane cellular proteins prior to a final chromatographic step or any analysis (e.g. mass- spectrometry, UV-vis absorption, circular dichroism measurements etc.).

The approach uses positively charged amines which bind to negative charges on the membrane (or any entity embedded or adsorbed to the membrane). This leads to generations of membrane aggregates that can be precipitated at low centrifugal force in comparison to independent membranes.

Specifically, the present inventors have demonstrated that oligo-amines, carrying from +3 to +6 positive charges, are able to function as water-soluble mediators for conjugating native membranes.

Membrane conjugation is efficient, reversible (upon addition of salt, e.g. NaCl), can be performed in buffered solutions within a wide range of pH values (7-2.5) and is correlated with the net charge of the oligo-amine used. As illustrated in Figures 8A-B and Figures 9A-F, membrane fragments from halophilic bacteria containing xanthrorhodopsin were successfully conjugated while preserving protein secondary structure as well as the native site of the chromophore. In addition, the present inventors successfully precipitated membrane fragments from halophilic bacteria containing bacteriorhodopsin (bR).

The mechanism described possesses a number of inherent advantages with respect to other protocols in the literature: (a) Preservation of the bilayer composition since charged oligo-amines are not expected to embed themselves in the membrane and are readily removed by dialysis (b) The absence of Ca²⁺, which is known to perturb membrane structure by, for example, strongly binding to the negatively charged leaflet of membranes and lead to T-cell activation (c) Spectroscopic measurements can be conveniently performed in the presence of oligo-amines as these do not contain an aromatic moiety (d) The rate of membrane conjugation can be regulated by the type and concentration of the oligo-amine used (e) Membrane clustering can be reversed by NaCl. (f) Oligo-amines are commercially available, highly pure and stable toward acidic or basic conditions.

Thus, according to a first aspect of the present invention there is provided a method of precipitating nanostructures out of a mixture of cellular components, said nanostructures having an outer surface comprising phospholipids, the method comprising contacting the mixture with an oligo-amine having a sufficient number of amine groups such that it ionically conjugates with said nanostructures, thereby precipitating nanostructures out of a mixture of cellular components.

As used herein, the term “nanostructure” refers to a particle having a diameter ranging from about 1 nm to about 1500 nm (e.g. from 1 nm to 1200 nm, from 1 nm to 1000 nm, from 1 nm to 800 nm, from 1 nm to 500 nm, from 1 nm to 400 nm, from 10 nm to 1000 nm, from 10 nm to 800 nm, from 10 nm to 500 nm, from 10 nm to 400 nm, from 100 nm to 1000 nm, from 100 nm to 800 nm, from 100 nm to 500 nm, from 100 nm to 400 nm). In certain embodiments, the nanostructure comprises a single particle or a cluster of particles. In certain embodiments, the nanostructure comprises a core nanoparticle and a coating.

At least part of the outer surface of the nanostructure of this aspect of the present invention comprises phospholipids. Preferably, the phospholipids are organized into a bilayer. In one embodiment, the phospholipids comprise negatively charged phospholipids. In another embodiment, the phospholipids comprise neutral phospholipids. In still another embodiment, the nanostructures comprise both negatively charged and neutral phospholipids. In still another embodiment, the phospholipids are not positively charged. Exemplary phospholipids that may be comprised in the nanostructure include, but are not limited to phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylcholine and sphingomyelin.

The nanostructures may also include other components. Examples of such other components includes, without being limited thereto, fatty alcohols, fatty acids, and/or cholesterol esters or any other agent which may affect the surface charge, the membrane fluidity and assist in the incorporation of the biologically active lipid into the lipid assembly. Examples of sterols include cholesterol, cholesterol hemi succinate, cholesterol sulfate, or any other derivatives of cholesterol. Preferred lipid assemblies according the invention include those which form a liposome.

According to one embodiment, the nanostructure comprises a cell or nuclear membrane fragment.

The phrase “cell or nuclear membrane fragments” refers to fragments of membranes which are no longer intact (i.e. have been disrupted) and which no longer fully enclose cellular contents. Preferably, the nanostructures comprising cell or nuclear (open or closed, i.e. spherical) membrane fragments have a length (or diameter) ranging between 20 nm - 1 pm, as measured by dynamic light scattering (DLS), electron microscopy (EM).

Typically, the cell or nuclear membrane fragments comprise membrane proteins attached thereto (e.g. embedded or traversing the membrane).

As used herein, the term “membrane protein” refers to a protein that is associated with a cell membrane.

According to one embodiment, the membrane protein is a transmembrane protein (e.g. a single transmembrane a-helix (bitopic membrane protein), a polytopic transmembrane a-helical protein or a polytopic transmembrane b-sheet protein).

According to another embodiment, the membrane protein is a peripheral membrane protein. Such proteins may interact with the cell membrane by an amphipathic a-helix parallel to the membrane plane (in-plane membrane helix); by a hydrophobic loop; by a covalently bound membrane lipid (lipidation); or by electrostatic or ionic interactions with membrane lipids (e.g. through a calcium ion).

The membrane protein may serve any function - e.g. a receptor, an ion pump, an ion channel or a carrier protein.

In one embodiment, the membrane protein is a retinyledene protein (e.g. channelrhodopsin, bacteriorhodopsin, halorhodopsin, or proteorhodopsin).

The halorhodopsin may be derived from any halobacteria (e.g. Natronomonas pharaonis).

According to another embodiment, the nanostructure comprises a synthetic particle including a liposome, a nanocapsule, a nanosphere or a nanocage.

As used herein and as recognized in the art, liposomes include any synthetic (i.e., not naturally occurring) structure composed of lipid bilayers, which enclose a volume. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. The liposomes may be prepared by any of the known methods in the art [Monkkonen, J. et ah, 1994, J. Drug Target, 2:299-308; Monkkonen, J. et ah, 1993, Calcif. Tissue Int., 53:139-145; Lasic D., Liposomes Technology Inc., Elsevier, 1993, 63- 105. (chapter 3); Winterhalter M, Lasic D, Chem Phys Lipids, 1993 September; 64(l-3):35-43]

The liposomes may be unilamellar or may be multilamellar. The liposomes may be fabricated from a single phospholipid or mixtures of phospholipids. The liposomes may also comprise other lipid materials such as cholesterol. For fabricating liposomes with a negative electrical surface potential, acidic phospho- or sphingo- or other synthetic-lipids may be used. Preferably, the lipids have a high partition coefficient into lipid bilayers and a low desorption rate from the lipid assembly. Exemplary phospholipids that may be used for fabricating liposomes with a negative electrical surface potential include, but are not limited to phosphatidylserine, phosphatidic acid, phosphatidylcholine and phosphatidyl glycerol.

According to still another embodiment, the nanostructure is an exosome.

As used herein, the term "exosome" refers to an extracellular vesicle that is released from a cell upon fusion of a multi vesicular body (MVB) with the plasma membrane.

The exosome may (a) have a size of between 30 nm (nanometer) and 120 nm (nanometer) as determined by electron microscopy; (b) comprises a complex of molecular weight >100 kDa (kilodalton), comprising proteins of <100 kDa; (c) comprises a complex of molecular weight >300 kDa, comprising proteins of <300 kDa; (d) comprises a complex of molecular weight >1000 kDa; (e) has a size of between 2 nm and 200 nm, as determined by filtration against a 0.2 pM filter and concentration against a membrane with a molecular weight cut-off of 10 kDa; or (f) a hydrodynamic radius of below 100 nm, as determined by laser diffraction or dynamic light scattering.

As mentioned, the nanostructures of this aspect of the present invention are precipitated out of a mixture of cellular components.

In another embodiment, the nanostructures are precipitated out of a mixture of non-cellular components.

In some embodiments, the mixture of cellular components is a cell lysate.

As used herein, the term "cell lysate" refers to an aqueous solution of cellular biological material, wherein a substantial portion of the cells of the cellular material have become disrupted and released their internal components.

In one embodiment, the cell lysate is prepared from whole cells. In another embodiment, the cell lysate may be prepared from a cellular organelle, such as a nuclear cell lysate.

In the case of a whole cell lysate, it will be appreciated that following cell membrane disruption, the cell lysate may be treated so as to remove organelles greater than about 2 microns (e.g. cell nucleii). Thus, for example the whole cell lysate may be centrifuged so as to precipitate cell nucleii from the cell lysate. Exemplary centrifugation conditions include 1-5 minutes at 500- 1000 x g (e.g. 2 minutes at 985 x g).

In one embodiment, the cell lysate is a whole cell lysate which comprises cell membrane fragments.

In another embodiment, the cell lysate is a nuclear lysate which comprises nuclear membrane fragments. The cell lysate may be prepared from any cell. The cells may be eukaryotic (e.g. mammalian, plant, fungus) or prokaryotic (bacteria).

The bacteria may be a gram positive or a gram negative bacteria.

The term "Gram-positive bacteria" as used herein refers to bacteria characterized by having as part of their cell wall structure peptidoglycan as well as polysaccharides and/or teichoic acids and are characterized by their blue-violet color reaction in the Gram-staining procedure. Representative Gram-positive bacteria include: Actinomyces spp., Bacillus anthracis, Bifidobacterium spp., Clostridium botulinum, Clostridium perfringens, Clostridium spp., Clostridium tetani, Corynebacterium diphtheriae, Corynebacterium jeikeium, Enterococcus faecalis, Enterococcus faecium, Erysipelothrix rhusiopathiae, Eubacterium spp., Gardnerella vaginalis, Gemella morbillorum, Leuconostoc spp., Mycobacterium abcessus, Mycobacterium avium complex, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium haemophilium, Mycobacterium kansasii, Mycobacterium leprae, Mycobacterium marinum, Mycobacterium scrofulaceum, Mycobacterium smegmatis, Mycobacterium terrae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Nocardia spp., Peptococcus niger, Peptostreptococcus spp., Proprionibacterium spp., Staphylococcus aureus, Staphylococcus auricularis, Staphylococcus capitis, Staphylococcus cohnii, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus lugdanensis, Staphylococcus saccharolyticus, Staphylococcus saprophyticus, Staphylococcus schleiferi, Staphylococcus similans, Staphylococcus warneri, Staphylococcus xylosus, Streptococcus agalactiae (group B streptococcus), Streptococcus anginosus, Streptococcus bovis, Streptococcus canis, Streptococcus equi, Streptococcus milleri, Streptococcus mitior, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes (group A streptococcus), Streptococcus salivarius, Streptococcus sanguis. The term "Gram-negative bacteria" as used herein refer to bacteria characterized by the presence of a double membrane surrounding each bacterial cell. Representative Gram-negative bacteria include Acinetobacter calcoaceticus, Actinobacillus actinomycetemcomitans, Aeromonas hydrophila, Alcaligenes xylosoxidans, Bacteroides, Bacteroides fragilis, Bartonella bacilliformis, Bordetella spp., Borrelia burgdorferi, Branhamella catarrhalis, Brucella spp., Campylobacter spp., Chalmydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Chromobacterium violaceum, Citrobacter spp., Eikenella corrodens, Enterobacter aerogenes, Escherichia cob, Flavobacterium meningosepticum, Fusobacterium spp., Haemophilus influenzae, Haemophilus spp., Helicobacter pylori, Klebsiella spp., Legionella spp., Leptospira spp., Moraxella catarrhalis, Morganella morganii, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multocida, Plesiomonas shigelloides, Prevotella spp., Proteus spp., Providencia rettgeri, Pseudomonas aeruginosa, Pseudomonas spp., Rickettsia prowazekii, Rickettsia rickettsii, Rochalimaea spp., Salmonella spp., Salmonella typhi, Serratia marcescens, Shigella spp., Treponema carateum, Treponema pallidum, Treponema pallidum endemicum, Treponema pertenue, Veillonella spp., Vibrio cholerae, Vibrio vulnificus, Yersinia enterocolitica, Yersinia pestis.

In another embodiment, the cells are archae cells, such as halobacterium cells.

Examples of halobacterium include Halobacterium salinarum, Haloferax denitrificans, Alorubrum distributum, Alobacterium salinarum, Halobacterium jilantaiense, Halorubrum lacusprofundi, Haloferax mediterranei, Halobacterium noricense, Natronomonas pharaonis, Halobacterium piscisalsi, Halorubrum saccharovoru, Halobacterium salinarum, Halorubrum sodomense, Halorubrum trapanicum, Haloarcula vallismortis and Halobacterium volcanii.

In one embodiment, the cells have been immortalized and are part of a cell line.

In another embodiment, the cells are part of a tissue preparation or an organism.

The cells may have been cultured (e.g. propagated) or taken directly from the cellular source without culturing.

The cell may be genetically modified so as to express the membrane protein. In another embodiment, the cell is not genetically modified.

There are a variety of ways to lyse cells. Well-known methods used include free-thawing, heat treatment, pressure treatment, mechanical grinding, sonication, treatment with chaotropes (e.g. guanidinium isothiocyante), non-ionic surfactants (e.g. Triton XI 00) and treatment with organic solvents (e.g. phenol).

It will be appreciated that the cell lysate is prepared such that the membrane proteins remain attached to (e.g. embedded in) the membrane and that non-membrane proteins do not become attached to the membrane during the process.

According to a particular embodiment the cell lysate is prepared without the use of chemical agents such as chaotropes or organic solvents.

As mentioned, the precipitation method of this aspect of the present invention is carried out by contacting the mixture of cellular components with an oligo-amine which can ionically bind to negatively charged phospholipids of the membrane. In some embodiments, the oligo-amine is capable of ionically binding to negatively charged groups on a protein which are embedded or bound to a membrane. In other embodiments the oligo-amine ionically binds to negatively charged phospholipids of the membrane and to negatively charged groups on a protein which is embedded or bound to the membrane. Preferably, the oligo-amine does not distort the structure of a protein to which it binds and/or cause distortion of the lipid membrane.

According to a particular embodiment, the oligo-amine has between 3 and 20 amine groups, more preferably between 3 and 9 amine groups and more preferably between 3 and 6 amine groups.

As used herein, the term “oligo-amine” refers to a compound having amino functional groups.

The oligo-amine of this aspect of the present invention comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amine groups.

Preferably, the molecular weight of the oligo-amine is between 100-2,000 Daltons.

The oligo-amine of this aspect of the present invention is typically non-aromatic oligo- amine.

In one embodiment, the oligo-amine is a linear oligo-amine (i.e. not ring shaped). The linear oligo-amine may be branched or not branched.

Linear oligo-amines contemplated by the present invention include, but are not limited to pentaethylenehexamine, tetraethylenepentamine pentahydrochloride and triethylenetetramine hydrate.

Other contemplated linear oligo-amines include bis(hexamethylene)triamine, N,N’N”trimethylbis(hexamethylene)triamine, 4-(aminomethyl)-l,8-octanediamine, triethylenetetramine, triethylenetetramine tetrahydrochloride, 1,1,4,7,10,10- hexamethyltriethylenetetramine, 1,4,7, 11-tetraazaundecane tetrahydrochloride, N,N-Bis(3- aminopropyl)-ethylenediamine, N,N-Bis(2-aminoethyl)-l, 3-propanediamine, N,N-Bis(3- aminopropyl)-l, 3-propanediamine, spermine, spermine tetrahydrochloride, tris(2- aminoethyl)amine and tetraethylenepentamine.

In another embodiment, the oligo-amine comprises a quaternary ammonium.

According to still another embodiment, the oligo-amine is a ring-shaped oligo-amine. Examples of such ring-shaped oligo-amines include, but are not limited to acetaldehyde ammonia trimer, formaldoxime trimer hydrochloride, 1,4,7-trizacyclononane, 1,4, 7-trimethyl- 1,4,7- trizacyclonane, 1,5,9-triazacyclododecane and 1, 5, 9-triazacyclododecane trihydrobromide.

The net positive charge of the oligo-amine is such that it is capable of binding (electrostatically) to either the negative charges of membrane protein amino acid residues and/or of membrane headgroups thus serving as mediators in the aqueous phase.

In one embodiment, the oligo-amine is not capable of embedding into the lipid bilayer of a cell membrane. The precipitation method of this aspect of the present invention is preferably carried out in the absence of detergents (or in a reaction which is substantially free of detergents).

The precipitation method of this aspect of the present invention is preferably carried out in the absence of precipitants ( e.g . PEG) and Ca²⁺ ions.

Following a sufficient incubation time, and under the appropriate pH conditions, the reactants form a complex. Typically, the pH of the mixture is maintained at an acidic pH (e.g. between 2.5-7, more preferably between 3-6.5) such that the oligo-amine carries at least 3, at least 4, at least 5, at least 6 or more positive charges.

Typically, the incubation is performed in the absence of a hydrophobic chelator (such as those disclosed in US Patent No. 10,030,224).

Once formed (seconds to hours), precipitation of the complex may be facilitated by centrifugation (e.g. ultra-centrifugation), although in some cases (for example, in the case of large complexes) centrifugation is not necessary or very mild centrifugation can be used (so at to render the solution more dense - e.g. for 1-5 minutes at a speed of 1000-3 OOOxg).

Optionally, following precipitation, if the nanostructure comprises a protein (e.g. a membrane protein), it may be purified.

Thus, according to another aspect of the present invention there is provided a method of purifying a membrane protein comprising:

(a) precipitating cell membrane fragments which comprise the membrane protein from a cell lysate according to the method described herein;

(b) isolating the cell membrane fragments following the precipitating; and

(c) solubilizing the membrane protein, thereby purifying the membrane protein.

As used herein the term “purifying” refers to at least separating the membrane protein from non-membrane proteins.

Isolation of the membrane fragments is typically effected by removal of the supernatant from the precipitated membrane fragments.

The oligo-amine may optionally be removed using methods known in the art including for example dialysis.

The membrane fragments may then be solublilized using methods known in the art.

The precipitated membrane fragments may be solubilized using a detergent.

In one embodiment, the detergent is a non-ionic detergent. Examples of non-ionic detergents include, but are not limited to decyl //-D-maltoside (DM), dodecyl //-D-maltoside (DDM), octyl //-D-glucoside (OG) and octyl //-D- l -thioglucoside (OTG). Depending on the intended use of the protein that is isolated and optionally solubilized, the protein may be subjected to further purification steps. This may be effected by using a number of biochemical methods which are well known in the art. Examples include, but are not limited to, fractionation on a hydrophobic interaction chromatography (e.g. on phenyl sepharose), ethanol precipitation, isoelectric focusing, reverse phase HPLC, chromatography on silica, chromatography on heparin sepharose, anion exchange chromatography, cation exchange chromatography, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography (e.g. using protein A, protein G, an antibody, a specific substrate, ligand or antigen as the capture reagent).

In one embodiment, the protein which is isolated is analyzed by spectroscopic measurements (either in the presence or absence of the oligo-amine).

In one embodiment, the protein which is isolated is crystallized.

As used herein the term “crystallizing” refers to the solidification of the molecule of interest so as to form a regularly repeating internal arrangement of its atoms and often external plane faces.

Several crystalization approaches which are known in the art can be applied to the sample in order to facilitate crystalization of the molecule of interest. Examples of crystallization approaches include, but are not limited to, the free interface diffusion method [Salemme, F. R. (1972) Arch. Biochem. Biophys. 151:533-539], vapor diffusion in the hanging or sitting drop method (McPherson, A. (1982) Preparation and Analysis of Protein Crystals, John Wiley and Son, New York, pp 82-127), and liquid dialysis (Bailey, K. (1940) Nature 145:934-935).

Presently, the hanging drop method is the most commonly used method for growing macromolecular crystals from solution; this approach is especially suitable for generating protein crystals. Typically, a droplet containing a protein solution is spotted on a cover slip and suspended in a sealed chamber that contains a reservoir with a higher concentration of precipitating agent. Over time, the solution in the droplet equilibrates with the reservoir by diffusing water vapor from the droplet, thereby slowly increasing the concentration of the protein and precipitating agent within the droplet, which in turn results in precipitation or crystallization of the protein.

In another embodiment, the protein is subjected to 2D gel electrophoresis.

The method of the present invention may be particularly useful for carrying out proteomic analysis of a membrane fraction of a cell. By removing non-relevant proteins from the sample prior to 2D gel electrophoresis analysis, the chance of eluting two proteins from a gel is decreased.

It will be appreciated that the oligo-amines may also be used to precipitate larger structures which comprise phospholipids. Thus, according to another aspect of the present invention there is provided a method of precipitating microstructures, said microstructures having an outer surface comprising phospholipids, the method comprising contacting said microstructures with an oligo-amine having a sufficient number of amine groups such that it ionically conjugates with said microstructures, thereby precipitating microstructures.

Exemplary contemplated microstructures comprise biological cells. In one embodiment, the cells are whole cells. In another embodiment, the cells are in a cell suspension (e.g. in a single cell suspension).

Oligo-amines of this aspect of the present invention are described herein above.

It is expected that during the life of a patent maturing from this application many relevant oligo-amines will be developed and the scope of the term oligo-amine is intended to include all such new technologies a priori.

As used herein the term “about” refers to ± 10 %.

The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

The term “consisting of’ means “including and limited to”.

The term "consisting essentially of' means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated herein above and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); “Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. T, ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

EXAMPLE 1

Conjugation of Native Membranes via Linear Oligo-amines

MATERIALS

Glycine, PEG-6000, NaCl, pentaethylenehexamine, tetraethylenepentamine pentahydrochloride, triethylenetetramine hydrate, diethylenetriamine, 2-Amino-2- (hydroxymethyl)-l, 3-propanediol (Tris), sodium phosphate (NaPi), sodium citrate, sodium acetate were obtained from Sigma-Aldrich (St. Louis, MO).

METHODS

Preparation of purple membranes: Halobacterium salinarum was grown from the S9 strain, and purple membranes containing bacteriorhodopsin were isolated as previously described.¹

Preparation of Xanthorhodopsin (XR) membranes: XR-membranes were prepared as described previously. ²

Bacteriorhodopsin (bR)-vesicle conjugation: Vesicles containing bR (bR-vesicles) were prepared according to Takeda et al ., ³. Freshly prepared bR vesicles were diluted (xl50) with DDW to a total volume of 1000 pL. 50 pL of the resulting dispersion were added to 50 pL of 10% PEG-6000 (w\v) plus 20 mM pentaethylenehexamine (pH 7) to a final volume of 100 pL. This mixture was vigorously vortexed and incubated for 45 min at 19 °C. 10 pL of the mixture was transferred onto an electron microscope grid and air dried at room temperature to complete dehydration.

Conjugation of purple membranes containing bacteriorhodopsin (bR) at pH 7: Purple membranes were diluted (xl50) with DDW to a total volume of 1000 pL. Diluted bR-membranes were mixed in a 1:1 ratio with a solution containing 100 mM Tris (pH 7) and 4 mM of either: pentaethylenehexamine; tetraethylenepentamine pentahydrochloride; triethylenetetramine hydrate or diethylenetriamine (all neutralized to pH 7 with either NaOH or HC1). For light microscopy imaging, 5 pL aliquots of the resulting mixture were placed on siliconized cover-slides and incubated over wells containing DDW in the dark at 19 °C on VDX™ crystallization plates (Hampton Research). For AFM and SEM analysis, 10 pL aliquots were placed on mica or silicon wafers and allowed to dry at room temperature.

Conjugation of purple membranes at pH 4 or 2.5: The protocol was identical to the one used at pH 7 except for the use of 50 mM AcOH (pH 4) or 50 mM Glycine (pH 2.5) instead of 50 mM Tris-HCl (pH 7).

Conjugation of membranes containing xanthrorhodopsin (XR) at pH 7: The protocol for conjugating XR-membranes was identical to the one described above for bR-membranes except for the need to dialyze XR-membranes prior to the addition of the oligo-amines due to the presence of salts in the XR-membrane preparation (see the dialysis protocol below). Therefore, XR- membranes were dialyzed twice for 1.5 hours against DDW at 19 °C. The resulting dialyzed XR- containing membranes were diluted (x5) with DDW to a total volume of 500 pL and used as such for further analysis.

Dialysis: XR-containing membranes (200 pL) were dialyzed three times against 50 mL DDW at 10 °C in the dark.

Circular dichroism (CD) spectroscopy: Freshly prepared bR membranes (4 pi) were mixed with 196 pi buffer containing 50 mM Tris pH 7 and 0.8 mM of either pentaethylenehexamine, tetraethylenepentamine pentahydrochloride, triethylenetetramine hydrate or diethylenetriamine. The resulting mixture was incubated for 5 minutes at room temperature and subjected to CD analysis using a Chirascan CD spectrometer (Applied Photophysics). CD spectra report ellipticity (Q), proportional to the difference in absorbance of left and right circularly polarized light [Q = 3300°] (AL-AR) as a function of wavelength. A quartz 1 x 1 cm path length cuvette was used. The CD spectra were recorded with 1 nm bandwidth resolution in 1 nm steps at 20 °C. The CD spectra were corrected for baseline distortion by subtracting a reference spectrum of the corresponding buffer.

Scanning electron microscopy (SEM): Freshly prepared samples without metal coating were characterized using the Tescan Ultra-High Resolution MAIA 3 FE-SEM. Beam voltage 5kV and In-Beam SE detector were used for samples shown in Figure 6 (B, C) and Figure 9 (A-F). In Figure 6 (A, D and E), the SE(BDM) detector was used. Beam voltages 5kV and 3kV were used for Figure 6 A and Figure 6 D, E, respectively. UV-vis spectroscopy: Absorption measurements were performed using HP 8453 UV-Vis spectrophotometer.

RESULTS

Spectroscopy of purple membranes: UV-Vis spectroscopy demonstrated that the characteristic absorption peak is not changed significantly in the presence of 20 mM of each of the oligo-amines studied when incubated at pH 7 (50 mM NaPi) and 19 °C for 24 hours in the dark (Figure 2A). These findings were encouraging: pairs of negatively charged amino acid residues of bR that are in close proximity to each other ( e.g . D36 & D38; D102 & D104; E232 & E234; E237 & D242²²), could theoretically interact strongly with the oligo-amines and distort the protein structure. Since no significant change in the absorption of the bR retinal chromophore at 568 nm was observed, it was concluded that its native structure had not been significantly perturbed by the addition of oligo-amines. Circular dichroism (CD) measurements were used to monitor the effects of the oligo-amines on bR secondary structure, dominated by the 7 trans-membrane a-helices. In (Figure 2B), CD spectra are presented. The negative band at -225 nm and the negative-going shoulder at -210 nm are characteristic of a-helix. These bands and their ratio are not affected by the addition of the oligo-amines. However, a small reduction of band amplitude is observed for the more heavily charged oligo-amines. This does not necessarily indicate perturbation of secondary structure, since “absorption flattening” is also well known for sample non-homogeneity due to large particle size. ²³

Light microscopy of purple membranes: Evidence for the long term stability of the conjugated purple membranes was obtained by following changes in the system using light microscopy at time points from 15 minutes to 5 weeks (sample kept in the dark at 19 °C) after sample preparation (Figure 3A). In the absence of oligo-amine, purple membranes were evenly dispersed even after 1 week ([C]). However, introduction of oligo-amines with +5 or +6 positive charges showed a clear effect after 15 min incubation at 19 °C. While the +5 analog led to inhomogeneous distribution of purple membranes, the +6 analog led to concentrated membranes that were confined within a single region with sharp boundaries towards the aqueous phase. By contrast, no change in system homogeneity was observed with the +3 or +4 oligo-amine analogs during the same time frame. After 1 week incubation, the sample containing the +6 analog was further compacted and exhibited an even deeper purple color; the +5 and +4 analogs induced further phase separation, while the +3 analog had no noticeable effect. Thus a clear correlation between the oligo-amine net charge (at pH 7) and the efficiency of membrane conjugation was observed. Nevertheless, preservation of the purple color after 5 weeks, in all samples containing the oligo-amines, demonstrated the non-denaturing characteristics of the membrane conjugation mechanism, as purple color is correlated with preservation of the native retinal binding site in bR. Consistent with the above, quantitative precipitation of purple membranes and formation of a dark purple pellet was observed (Figure 3B), shortly after the +6 oligo-amine was introduced into the system at neutral pH (20 mM NaPi), whereas in its absence, purple membranes were homogenously distributed.

NaCl reverses conjugation of purple membranes: As expected for electrostatic interactions, we observed that membrane conjugation can be reversed by a relatively large concentration of salt. Membrane clusters that were initially generated with +6 or +5 oligo-amines dissociated completely upon addition of NaCl (Figures 4A-B). It was found that a 15 min incubation with 1 M NaCl (in the dark and at 19 °C) was sufficient to achieve a relatively homogeneous distribution of purple membrane fragments initially conjugated via the +6 oligo- amine (Figure 4A) whereas only 0.1 M NaCl was needed to observe similar results with purple membranes conjugated via the +5 analog (Figure 4B). This tenfold difference in salt concentration is probably due to the greater binding affinity of the +6 analog to the protein, the phospholipid anionic head groups, or both, in comparison to the +5 derivative. It also explains the longer incubation times (e.g. 3 hr), required to improve sample homogeneity with membranes conjugated via the +6 analog (Figure 4A).

Effect of acidic pH on conjugation of purple membranes: To support the suggestion concerning the role of binding affinity, membrane clustering was studied at acidic pH values that would reduce the net negative charge of the anionic phospholipids and of bR and thus increase the efficiency of membrane clustering with oligo-amines with fewer positive charges (Figure 5). Indeed, at pH 4, the +3 analog efficiently conjugated purple membranes while in its absence, purple membranes were homogeneously distributed (Figure 5). Interestingly, no major differences with respect to process efficiency were observed between the +3 and +6 analogs at pH 4 as both led to highly concentrated purple membranes with defined boundaries (Figure 5). Similar conjugation was also observed at pH 2.5 with both oligo-amine derivatives (Figure 5). The observed color change of purple membranes, from purple to blue, under highly acidic conditions (Figure 5, [C]) is known to occur below pH 3 due to protonation of a carboxylate (Asp85) located in the retinal chromophore binding site. ^24-27 Moreover, the blue color of purple membranes was formed following membranes deionization, and was reverted back to the original purple color once cations (e.g. Na⁺, K⁺, Mg²⁺, Mn²⁺, Pb²⁺) were added ^26-28 to the medium or positively charged polyelectrolytes (e.g. diethyl aminoethyl dextran). ²⁹ This explains why samples containing the +3 or +6 oligo-amines regenerated the purple color of the membranes (Figure 5). SEM images of purple membrane conjugation : Scanning electron microscopy (SEM) was used to provide higher resolution images of the system before and after oligo-amine addition. As expected, correlation between the net charge of the oligo-amine and the size of the resulting membrane aggregates was observed (Figures 6A-E). In the absence of any oligo-amine, purple membrane fragments (0.3-2 pm) were randomly distributed (Figure 6A). However, the +3 derivative was sufficient to generate membrane clusters (Figure 6B) that reached mm diameter with the +6 analog (Figure 6E). These findings support the contention that the longer the oligo- amine and the greater the number of positive charges on the water-soluble mediator, membrane clustering is more efficient and accordingly clusters increase in size. It was further noted that vesicles prepared from purple membranes according to Takeda et al., ³ were observed to adhere to each other and to form dimers and trimers (Figures 7A-B) in the presence of the +6 oligo-amine analog, while in its absence, no clustering or (possible) fusion was observed (not shown).

Conjugation of cell membranes of eubacterium salinibacter ruber: To assess the potential generality of the membrane conjugation protocol, the present inventors applied it to native membrane fragments containing a second intrinsic membrane protein (MP), Xanthorhodopsin. Xanthorhodopsin (XR) ³⁰ is a retinal -based proton pump in the cell membranes of the halophilic eubacterium Salinibacter ruber. ³¹ XR exhibits high homology to the bR protein; however, in addition to the covalently bound all -trans retinal chromophore, through a protonated Schiff-base to Lys-240, it also contains a carotenoid (Salinixanthin), which acts as a light harvesting antenna by providing additional excitation energy for retinal isomerization and proton transport. ^{3 32-34} Accordingly, the effect of the +6 oligo-amine was studied on the characteristic absorption of XR (kmax = 492 nm). No significant change in XR absorption was observed (Figure 8A). Moreover, the +6 analog demonstrated its ability to efficiently precipitate XR-membranes at pH 7 and to generate a reddish pellet after brief centrifugation (Figure 8B). SEM analysis was used again to image the system after addition of each of the 4 oligo-amines (Figures 9A-F). As expected, in the absence of the oligo-amines, XR-membrane fragments (0.1 - 0.3 pm) were randomly dispersed (Figure 9A) and membrane clustering was found to be correlated with the oligo-amine net charge (Figure 9B-F). Moreover, only in the presence of the +6 analog, some evidence for fusion of XR-membranes was obtained, producing a tree leaf type architecture (Figure 9E). EXAMPLE 2

Purification of bacteriorhodopsin (bR) with pentaethylenehexamine (PEHA)

MATERIALS AND METHODS

Purification of bR, embedded within purple membranes from the E. coli lysate (serving as an artificial background), was accomplished with the oligo-amine: pentaethylenehexamine (PEHA) at neutral pH. Purification was initiated by the addition of PEHA (4 mM, pH 7) to a mixture of purple membranes and the lysate (50 pL) in 25 mM Tris pH 7. After 1 minute of incubation at room temperature, a short spin was applied (376xg-, 5 minutes), the supernatant discarded and the resulting pellet was washed twice with DDW (100 pL) using higher centrifugation force (14K, 2 minutes).

RESULTS

The chemical composition of the washed pellet is shown in Figure 10 lane 7. The flow through in the absence of the oligo-amine (PEHA) (lane 4), contains significantly more bR in comparison to the flow-through in the presence of PEHA (lane 6). This observation fits well with the ability of the oligo-amine to efficiently conjugate bR membranes and precipitate the latter, thereby excluding them from the supernatant (i.e. the flow-through sample). Consistent with the above is the greater recovery yields of bR in the presence of PEHA (lane 7 vs. lane 5). The purity of the recovered membrane protein (lane 7) is very similar to that present in the stock solution (lane 2).

REFERENCES

1. Oesterhelt, D.; Stoeckenius, W., Isolation of the cell membrane of Halobacterium halobium and its fractionation into red and purple membrane. Methods in enzymology 1974, 31, 667-78. 2. Balashov, S. P.; Imasheva, E. S.; Boichenko, V. A.; Anton, J.; Wang, J. M.; Lanyi, J. K.,

Xanthorhodopsin: a proton pump with a light-harvesting carotenoid antenna. Science (New York, N.Y.) 2005, 309 (5743), 2061-4.

3. Takeda, K.; Sato, H.; Hino, T.; Kono, M.; Fukuda, K.; Sakurai, I.; Okada, T.; Kouyama, T., A novel three-dimensional crystal of bacteriorhodopsin obtained by successive fusion of the vesicular assemblies. Journal of molecular biology 1998, 283 (2), 463-74.

4. Singer, S. J.; Nicolson, G. L., The fluid mosaic model of the structure of cell membranes. Science (New York, N.Y.) 1972, 175 (4023), 720-31.

5. Supported Lipid Bilayers. In Wiley Encyclopedia of Chemical Biology, pp 1-12.

6. Tamm, L. K.; McConnell, H. M., Supported phospholipid bilayers. Biophysical journal 1985, 47 (1), 105-13.

7. Seu, K. J.; Pandey, A. P.; Haque, F.; Proctor, E. A.; Ribbe, A. E.; Hovis, J. S., Effect of Surface Treatment on Diffusion and Domain Formation in Supported Lipid Bilayers. Biophysical journal 2007, 92 (7), 2445-2450. 8. Kiessling, V.; Crane, J. M.; Tamm, L. K., Transbilayer effects of raft-like lipid domains in asymmetric planar bilayers measured by single molecule tracking. Biophysical journal 2006, 91 (9), 3313-26.

9. Tamm, L. K.; Tatulian, S. A., Infrared spectroscopy of proteins and peptides in lipid bilayers. Quarterly reviews of biophysics 1997, 30 (4), 365-429.

10. Merzlyakov, M.; Li, E.; Casas, R.; Hristova, K., Spectral Forster resonance energy transfer detection of protein interactions in surface-supported bilayers. Langmuir : the ACS journal of surfaces and colloids 2006, 22 (16), 6986-92.

11. Thompson, N. L.; Steele, B. L., Total internal reflection with fluorescence correlation spectroscopy. Nature protocols 2007, 2 (4), 878-90.

12. Hinterdorfer, P.; Baber, G.; Tamm, L. K., Reconstitution of membrane fusion sites. A total internal reflection fluorescence microscopy study of influenza hemagglutinin-mediated membrane fusion. The Journal of biological chemistry 1994, 269 (32), 20360-8.

13. Fix, M.; Melia, T. J.; Jaiswal, J. K.; Rappoport, J. Z.; You, D.; Sollner, T. FL; Rothman, J. E.; Simon, S. M., Imaging single membrane fusion events mediated by SNARE proteins. Proceedings of the National Academy of Sciences of the United States of America 2004, 101 (19), 7311-6.

14. Cornell, B. A.; Braach-Maksvytis, V. L. B.; King, L. G.; Osman, P. D. J.; Raguse, B.; Wieczorek, L.; Pace, R. J., A biosensor that uses ion-channel switches. Nature 1997, 387 (6633), 580-583.

15. Stelzle, M.; Weissmueller, G.; Sackmann, E., On the application of supported bilayers as receptive layers for biosensors with electrical detection. The Journal of Physical Chemistry 1993, 97 (12), 2974-2981.

16. Stora, T.; Lakey, J. FL; Vogel, FL, Ion-Channel Gating in Transmembrane Receptor Proteins: Functional Activity in Tethered Lipid Membranes. Angewandte Chemie (International ed in English) 1999, 38 (3), 389-392.

17. Patchomik, G.; Namboothiri, I. N.; Nair, D. K.; Wachtel, E.; Cohen, S. R.; Friedman, N.; Sheves, M., Engineered-membranes: a novel concept for clustering of native lipid bilayers. Journal of colloid and interface science 2012, 388 (1), 300-5.

18. Garavito, R. M.; Ferguson-Miller, S., Detergents as tools in membrane biochemistry. The Journal of biological chemistry 2001, 276 (35), 32403-6.

19. Luecke, FL; Schobert, B.; Richter, H. T.; Cartailler, J. P.; Lanyi, J. K., Structure of bacteriorhodopsin at 1.55 A resolution. Journal of molecular biology 1999, 291 (4), 899-911.

20. Palsdottir, H.; Hunte, C., Lipids in membrane protein structures. Biochimica et Biophysica Acta (BBA) - Biomembranes 2004, 1666 (1), 2-18.

21. Adamian, L.; Naveed, H.; Liang, J., Lipid-binding surfaces of membrane proteins: evidence from evolutionary and structural analysis. Biochimica et biophysica acta 2011, 1808 (4), 1092-1102.

22. Khorana, H. G.; Gerber, G. E.; Herlihy, W. C.; Gray, C. P.; Anderegg, R. J.; Nihei, K.; Biemann, K., Amino acid sequence of bacteriorhodopsin. Proceedings of the National Academy of Sciences of the United States of America 1979, 76 (10), 5046-50.

23. Miles, A. J.; Wallace, B. A., Circular dichroism spectroscopy of membrane proteins. Chemical Society reviews 2016, 45 (18), 4859-4872.

24. Kimura, Y.; Ikegami, A.; Stoeckenius, W., SALT AND pH-DEPENDENT CHANGES OF THE PURPLE MEMBRANE ABSORPTION SPECTRUM. Photochemistry and Photobiology 1984, 40 (5), 641-646.

25. Moore, T. A.; Edgerton, M. E.; Parr, G.; Greenwood, C.; Perham, R. N., Studies of an acid- induced species of purple membrane from Halobacterium halobium. The Biochemical journal 1978, 171 (2), 469-76. 26. Mowery, P. C.; Lozier, R. H.; Chae, Q.; Tseng, Y. W.; Taylor, M.; Stoeckenius, W., Effect of acid pH on the absorption spectra and photoreactions of bacteriorhodopsin. Biochemistry 1979, 75 (19), 4100-7.

27. Fischer, U.; Oesterhelt, D., Chromophore equilibria in bacteriorhodopsin. Biophysical journal 1979, 25 (2), 211-30.

28. Edgerton, M. E.; Moore, T. A.; Greenwood, C., Salt reversal of the acid-induced changes in purple membrane from Halobacterium halobium. FEBS letters 1978, 95 (1), 35-9.

29. Bakker-Grunwald, T.; Hess, B., Interactions of bacteriorhodopsin-containing membrane systems with polyelectrolytes. The Journal of membrane biology 1981, 60 (1), 45-49.

30. Boichenko, V. A.; Wang, J. M.; Anton, T; Lanyi, J. K.; Balashov, S. P., Functions of carotenoids in xanthorhodopsin and archaerhodopsin, from action spectra of photoinhibition of cell respiration. Biochimica et biophysica acta 2006, 1757 (12), 1649-56.

31. Anton, J.; Oren, A.; Benlloch, S.; Rodriguez -Valera, F.; Amann, R.; Rossello-Mora, R., Salinibacter ruber gen. nov., sp. nov., a novel, extremely halophilic member of the Bacteria from saltern crystallizer ponds. International journal of systematic and evolutionary microbiology 2002, 52 (Pt 2), 485-91.

32. Slouf, V.; Balashov, S. P.; Lanyi, J. K.; Pullerits, T.; Polivka, T., Carotenoid response to retinal excitation and photoisomerization dynamics in xanthorhodopsin. Chemical physics letters 2011, 516 (1-3), 96-101.

33. Gdor, T; Zhu, J.; Loevsky, B.; Smolensky, E.; Friedman, N.; Sheves, M.; Ruhman, S., Investigating excited state dynamics of salinixanthin and xanthorhodopsin in the near-infrared. Physical Chemistry Chemical Physics 2011, 13 (9), 3782-3787.

34. Zhu, J.; Gdor, T; Smolensky, E.; Friedman, N.; Sheves, M.; Ruhman, S., Photoselective ultrafast investigation of xanthorhodopsin and its carotenoid antenna salinixanthin. The journal of physical chemistry. B 2010, 114 (8), 3038-45.

35. Pedersen, U. R.; Leidy, C.; Westh, P.; Peters, G. H., The effect of calcium on the properties of charged phospholipid bilayers. Biochimica et Biophysica Acta (BBA) - Biomembranes 2006, 1758 (5), 573-582.

36. Tadross, M. R.; Tsien, R. W.; Yue, D. T., Ca2+ channel nanodomains boost local Ca2+ amplitude. Proceedings of the National Academy of Sciences of the United States of America 2013, 770 (39), 15794-9.

37. Shi, X.; Bi, Y.; Yang, W.; Guo, X.; Jiang, Y.; Wan, C.; Li, L.; Bai, Y.; Guo, J.; Wang, Y.; Chen, X.; Wu, B.; Sun, H.; Liu, W.; Wang, J.; Xu, C., Ca2+ regulates T-cell receptor activation by modulating the charge property of lipids. Nature 2013, 493 (7430), 111-5.

38. Fu, Q.; Piai, A.; Chen, W.; Xia, K.; Chou, J. J., Structure determination protocol for transmembrane domain oligomers. Nature protocols 2019, 14 (8), 2483-2520.

39. Diller, A.; Loudet, C.; Aussenac, F.; Raffard, G.; Fournier, S.; Laguerre, M.; Grelard, A.; Opella, S. J.; Marassi, F. M.; Dufourc, E. J., Bicelles: A natural 'molecular goniometer' for structural, dynamical and topological studies of molecules in membranes. Biochimie 2009, 91 (6), 744-751.

40. Ujwal, R.; Bowie, J. U., Crystallizing membrane proteins using lipidic bicelles. Methods (San Diego, Calif) 2011, 55 (4), 337-341.

41. Nasr, M. L.; Baptista, D.; Strauss, M.; Sun, Z.-Y. J.; Grigoriu, S.; Huser, S.; Pliickthun, A.; Hagn, F.; Walz, T.; Hogle, J. M.; Wagner, G., Covalently circularized nanodiscs for studying membrane proteins and viral entry. Nature methods 2016, 14, 49.

42. Nikolaev, M.; Round, E.; Gushchin, T; Polovinkin, V.; Balandin, T.; Kuzmichev, P.; Shevchenko, V.; Borshchevskiy, V.; Kuklin, A.; Round, A.; Bernhard, F.; Willbold, D.; Biildt, G.; Gordeliy, V., Integral Membrane Proteins Can Be Crystallized Directly from Nanodiscs. Crystal Growth & Design 2017, 77 (3), 945-948.

43. Mio, K.; Sato, C., Lipid environment of membrane proteins in cryo-EM based structural analysis. Biophys Rev 2018, 10 (2), 307-316. Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is/are hereby incorporated herein by reference in its/their entirety.

Claims

WHAT IS CLAIMED IS:

1. A method of precipitating nanostructures out of a mixture of cellular components, said nanostructures having an outer surface comprising phospholipids, the method comprising contacting the mixture with an oligo-amine having a sufficient number of amine groups such that it ionically conjugates with said nanostructures, thereby precipitating nanostructures out of a mixture of cellular components.

2. The method of claim 1, wherein said oligo-amine comprises between 3 and 20 amine groups.

3. The method of claim 1, wherein said oligo-amine comprises between 3 and 6 amine groups.

4. The method of any one of claims 1-3, wherein said outer surface of said nanostructures comprise a protein.

5. The method of any one of claims 1-4, wherein said nanostructures comprise cell membrane fragments and said mixture of cellular components is a cell lysate.

6. The method of claim 5, wherein said membrane fragments comprise a membrane protein.

7. The method of any one of claims 1-4, wherein said nanostructures comprise liposomes.

8. The method of any one of claims 1-4, wherein said nanostructures are synthetic nanostructures.

9. The method of any one of claims 1-4, wherein said nanostructures comprise exosomes.

10. The method of any one of claims 1-9, wherein said oligo-amine has a molecular weight below 2,000 Daltons.

11. The method of any one of claims 1-10, wherein said oligo-amine is a non-aromatic oligo-amine.

12. The method of any one of claims 1-11, wherein said oligo-amine is a linear oligo- amine.

13. The method of claim 12, wherein said linear oligo-amine is selected from the group consisting of pentaethylenehexamine, tetraethylenepentamine pentahydrochloride and triethylenetetramine hydrate.

14. The method of any one of claims 1-11 wherein said oligo-amine comprises a quaternary ammonium.

15. The method of any one of claims 1-11, wherein said oligo-amine is a ring-shaped oligo-amine.

16. The method of any one of claims 5, 6 or 10-15, wherein said cell membrane fragments are generated by sonicating whole cells.

17. The method of any one of claims 5, 6 or 10-15, wherein said cell lysate is a whole cell lysate.

18. The method of any one of claims 5, 6 or 10-15, wherein said cell lysate is devoid of organelles greater than about 2 microns.

19. The method of any one of claim 1-18, further comprising isolating said nanostructures from said mixture following said precipitating.

20. The method of claim 19, wherein further comprising solubilizing proteins of said cell membrane fragments following said isolating.

21. The method of claim 20, wherein said solubilizing is effected with a detergent.

22. The method of claim 20, wherein said detergent comprises a non-ionic detergent.

23. The method of claim 21, wherein said detergent is selected from the group consisting of decyl //-D-maltoside (DM), dodecyl //-D-maltoside (DDM), octyl //-D-glucoside (OG) and octyl //-D- l -thioglucoside (OTG).

24. The method of any one of claims 1-19, wherein said contacting is effected in the absence of a detergent.

25. The method of any one of claims 1-19, wherein said contacting is effected in the absence of a hydrophobic chelator.

26. The method of claims 5 or 6, wherein said cell membrane fragments have a length ranging between 200-700nm.

27. The method of claims 5 or 6, wherein said cell lysate is derived from a bacterial cell.

28. The method of claims 5 or 6, wherein said cell lysate is derived from an archaeal cell.

29. The method of claim 28, wherein said archaeal cell is a halobacterium cell.

30. The method of claim 29, wherein said halobacterium is selected from the group consisting of Halobacterium salinarum, Haloferax denitrificans, Alorubrum distributum, Alobacterium salinarum, Halobacterium jilantaiense, Halorubrum lacusprofundi, Haloferax mediterranei, Halobacterium noricense, Natronomonas pharaonis, Halobacterium piscisalsi, Halorubrum saccharovoru, Halobacterium salinarum, Halorubrum sodomense, Halorubrum trapanicum, Haloarcula vallismortis and Halobacterium volcanii.

31. A method of purifying a membrane protein comprising:

(a) precipitating cell membrane fragments which comprise the membrane protein from a cell lysate according to the method of claim 6;

(b) isolating said cell membrane fragments following said precipitating; and

(c) solubilizing said membrane protein, thereby purifying the membrane protein.

32. The method of claim 31, wherein said solubilizing is effected using a detergent.

33. The method of claim 32, wherein said detergent is a non-ionic detergent.

34. The method of claim 32, wherein said detergent is selected from the group consisting of decyl //-D-maltoside (DM), dodecyl //-D-maltoside (DDM), octyl //-D-glucoside (OG) and octyl //-D- l -thioglucoside (OTG).

35. The method of claim 31, wherein said isolating comprises:

(a) centrifuging said cell lysate to form a pellet comprising said cell membrane fragments and a supernatant; and

(b) removing said supernatant from said pellet.

36. The method of claim 31, wherein the membrane protein is a retinyledene protein.

37. The method of claim 36, wherein said retinyledene protein is selected from the group consisting of channelrhodopsin, bacteriorhodopsin, halorhodopsin, and proteorhodopsin.

38. A method of precipitating microstructures having an outer surface comprising phospholipids, the method comprising contacting the microstructures with an oligo-amine having a sufficient number of amine groups such that it ionically conjugates with said nanostructures, thereby precipitating microstructures.

39. The method of claim 38, wherein said oligo-amine comprises between 3 and 20 amine groups.

40. The method of claim 38, wherein said oligo-amine comprises between 3 and 6 amine groups.

41. The method of claim 38, wherein said microstructures comprise biological cells.