Methods of generating supramolecular structures

Abstract

A method of generating a supramolecular structure is disclosed.The method comprises (a) contacting a divalent or trivalent metal cation with at least one protein-based molecule comprising at least two histidine tags under conditions that generate the supramolecular structure; and (b) isolating the supramolecular structure. Uses of the supramolecular structures are also disclosed.

Description

[0001] METHODS OF GENERATING SUPRAMOLECULAR STRUCTURES

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 575,819, filed April 7, 2024, the contents of which are incorporated herein by reference in their entirety.

[0004] SEQUENCE LISTING STATEMENT

[0005] The xml file, entitled 103179.xml, created on 6 April 2025, comprising 13,399 bytes, submitted concurrently with the filing of this application is incorporated herein by reference.

[0006] FIELD AND BACKGROUND OF THE INVENTION

[0007] The present invention, in some embodiments thereof, relates to methods of generating supramolecular structures of proteinaceous molecules using histidine tags and coordinating metal ions.

[0008] Higher-ordered protein assemblies are complexes made up of multiple protein molecules that come together to form larger, functional structures. These assemblies go beyond individual proteins or simple dimers / trimers — they can be massive, highly organized structures with specific biological roles.

[0009] Whilst there are examples in nature of fibrous proteins made of elongated polypeptide chains (e.g. collagens, elastins and silks), there are few examples of naturally occurring 2D protein arrays. These include (i) the purple membranes of halobacterial species composed of hexagonal arrays of tightly packed bacteriorhodopsin trimers surrounded by lipids; (ii) bacterial S-layer exoskeleton proteins found in arachaea and many members of bacteria, (iii) gap-junction plaques present in muscle and heart, and (iv) water-channels.

[0010] Higher-ordered protein assemblies have been generated using amphiphilic low molecular weight proteins (e.g. oleosin and hydrophobin) or by altering the protein’s native sequence so as to enhance maximal protein-protein interaction via the ideal layer-group of the target homooligomer.

[0011] Non-protein polymeric precursors terminated with metal-binding groups, including histidine or catechol, have been observed to form hydrogels in the presence of metal ions such as Zn2+, Fe2+and Fe3+[Fullenkamp, D.E., et al., Macromolecules, 2013. 46(3): p. 1167-1174; Holten- Andersen, N., et al., Proc Natl Acad Sci U S A, 2011. 108(7): p. 2651-; Mozhdehi, D., et al., J Am Chem Soc, 2014. 136(46): p. 16128-31], Additional art includes Weber et al., Nature Materials, Vol. 15, January 2016, page 13-26; Evers, T.H., et al., Protein Eng Des Sei, 2008. 21(8): p. 529-36; and Dong et al., Nano Letters (2009) 9, 2246-2250.

[0012] SUMMARY OF THE INVENTION

[0013] According to an aspect of the present invention there is provided a method of generating a supramolecular structure comprising:

[0014] (a) contacting a divalent or trivalent metal cation with at least one protein-based molecule comprising at least two histidine tags under conditions that generate the supramolecular structure; and

[0015] (b) isolating the supramolecular structure.

[0016] According to embodiments of the invention, the supramolecular structure is a nanofiber or microfiber.

[0017] According to embodiments of the invention, the supramolecular structure is a nanosheet or microsheet.

[0018] According to embodiments of the invention, the supramolecular structure is not a cyclic structure.

[0019] According to embodiments of the invention, the metal cation is selected from the group consisting of Zinc (Zn2+), Nickel (Ni2+), iron (Fe2+or Fe3+), or cobalt (Co2+).

[0020] According to embodiments of the invention, the divalent metal cation are Zinc (Zn2+) or Nickel (Ni2+).

[0021] According to embodiments of the invention, the protein-based molecule is a monomeric polypeptide.

[0022] According to embodiments of the invention, the protein-based molecule is a dimer or trimer.

[0023] According to embodiments of the invention, the first histidine tag of the at least two histidine tags is positioned at the N-terminus of the polypeptide and a second histidine tag of the at least two histidine tags is positioned at the C-terminus of the polypeptide.

[0024] According to embodiments of the invention, each of the at least two histidine tags comprise at least 4 consecutive histidines.

[0025] According to embodiments of the invention, the at least one protein-based molecule is water soluble. According to embodiments of the invention, the at least one protein-based molecule is a membrane protein.

[0026] According to embodiments of the invention, the at least one protein-based molecule is a fluorescent polypeptide.

[0027] According to embodiments of the invention, the at least one protein-based molecule is an enzyme or receptor.

[0028] According to embodiments of the invention, the at least one protein-based molecule is devoid of an amino acid sequence as set forth in SEQ ID NO: 8.

[0029] According to embodiments of the invention, the at least one protein-based molecule comprises at least two protein-based molecules, each comprising the at least two histidine tags.

[0030] According to embodiments of the invention, one of the at least two non-identical proteinbased molecule is an enzyme label and a second of the at least two non-identical protein-based molecule binds specifically to an Fc domain of an antibody.

[0031] According to embodiments of the invention, the second protein-based molecule is Protein G or Protein A.

[0032] According to another aspect of the invention, there is provided a supramolecular structure generated according to the method described herein.

[0033] According to another aspect of the invention, there is provided a composition comprising a purified supramolecular structure which comprises at least two repeating units, each repeating unit being represented by the formula P-D-P-D, wherein

[0034] P is a protein-based molecule comprising at least two histidine tags; and

[0035] D is a divalent or trivalent ion, wherein each protein-based molecule in the structure is non-covalently bound to at least one other protein-based molecule via the divalent or trivalent ion.

[0036] According to embodiments of the invention, the at least one the divalent or trivalent ion of the structure coordinates non-covalent binding of 2-6 the protein-based molecules.

[0037] According to embodiments of the invention, the at least one the divalent or trivalent ion of the structure coordinates non-covalent binding of four the protein-based molecules.

[0038] According to embodiments of the invention, the supramolecular structure is a nanofiber, a microfiber, a nanosheet or a microsheet.

[0039] According to embodiments of the invention, the composition is essentially devoid of structures composed of P-D-P.

[0040] According to embodiments of the invention, the metal cation is selected from the group consisting of Zinc (Zn2+), Nickel (Ni2+), iron (Fe2+or Fe3+), or cobalt (Co2+). According to embodiments of the invention, the metal cation are Zinc (Zn2+) or Nickel (Ni2+).

[0041] According to embodiments of the invention, the at least one protein based molecule is a polypeptide monomer.

[0042] According to embodiments of the invention, the at least one protein-based molecule is a dimer or trimer.

[0043] According to embodiments of the invention, a first histidine tag of the at least two histidine tags is positioned at the N-terminus of the polypeptide monomer and a second histidine tag of the at least two histidine tags is positioned at the C-terminus of the polypeptide monomer.

[0044] According to embodiments of the invention, each of the at least two histidine tags comprise at least 4 consecutive histidines.

[0045] According to embodiments of the invention, the protein-based molecule is water soluble.

[0046] According to embodiments of the invention, the protein-based molecule is a fluorescent polypeptide.

[0047] According to embodiments of the invention, the protein-based molecule is a biomolecule binding protein.

[0048] According to embodiments of the invention, the protein-based molecule is not an amyloid protein.

[0049] According to embodiments of the invention, the protein-based molecule is not an amphiphilic protein.

[0050] According to embodiments of the invention, the protein-based molecules of the supramolecular structure are not connected via amphiphilic peptides.

[0051] According to embodiments of the invention, the at least one protein-based molecule is an enzyme.

[0052] According to embodiments of the invention, the at least one protein-based molecule is a receptor.

[0053] According to embodiments of the invention, the composition is composed of at least two non-identical protein-based molecules, each comprising the at least two histidine tags.

[0054] According to embodiments of the invention, one of the at least two non-identical proteinbased molecules is an enzyme label and a second of the at least two non-identical protein-based molecules binds specifically to an FC domain of an antibody.

[0055] According to embodiments of the invention, the second protein-based molecule is Protein G or Protein A or a fragment thereof. According to yet another aspect of the invention, there is provided a method of isolating a biomolecule from a solution comprising:

[0056] (a) contacting the biomolecule with the composition of claim 33 under conditions that allows binding of the biomolecule to the biomolecule binding protein; and

[0057] (b) removing the supramolecular structure from the solution.

[0058] According to yet another aspect of the invention, there is provided a biomolecule affinity agent comprising a supramolecular structure which comprises at least two repeating units, each repeating unit being represented by the formula P-D-P-D, wherein

[0059] P is a detectable protein-based molecule comprising at least two histidine tags; and

[0060] D is a divalent or trivalent ion, wherein at least one detectable protein-based molecule in the structure is non-covalently bound to a second detectable protein-based molecule via the divalent or trivalent ion, wherein the supramolecular structure further comprises a protein that binds directly or indirectly to the biomolecule, the protein being non-covalently bound to the detectable proteinbased molecule via the divalent or trivalent ion.

[0061] According to embodiments of the invention, the protein binds to an FC region of an antibody.

[0062] According to embodiments of the invention, a ratio of the detectable protein-based molecule: the protein is at least 3:1.

[0063] According to yet another aspect of the invention, there is provided a method of detecting a biomolecule comprising:

[0064] (a) contacting the biomolecule with the biomolecule affinity agent described herein, under conditions which allow binding of the biomolecule to the biomolecule affinity agent; and

[0065] (b) detecting the detectable protein-based molecule.

[0066] According to embodiments of the invention, the method further comprises contacting the biomolecule with an antibody prior to step (a).

[0067] According to yet another aspect of the invention, there is provided a article of manufacture comprising a solid surface attached to the composition described herein.

[0068] According to embodiments of the invention, the article of manufacture is a medical device.

[0069] According to embodiments of the invention, the article of manufacture is a detection device.

[0070] According to yet another aspect of the invention, there is provided a method of performing an enzymatic reaction comprising contacting the composition described herein with starting components of an enzymatic reaction under conditions that allow the enzyme to catalyze the enzymatic reaction, thereby performing the enzymatic reaction.

[0071] According to yet another aspect of the invention, there is provided a method of generating an antibody in a subject comprising administering to the subject the composition described herein, thereby generating the antibody.

[0072] According to embodiments of the invention, the composition is devoid of an additional adjuvant.

[0073] 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.

[0074] BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0075] 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.

[0076] In the drawings:

[0077] FIGs. 1A-B: Cartoon of hexa-His ([Hise]) ligand - nickel (Figure 1A) or zinc (Figure IB) conjugation with the potential of promoting self-assembly of protein fibers or sheets as macromolecular biopolymers. [Hise]-tags are added to expression genes in E. coli BL21(DE3) competent cells at both the N- and C-terminal ends of the proteins. Dimensions are not to scale.

[0078] FIGs. 2A-D. Figure 2A. Cryo-TEM imaging of fibrous protein in vitreous ice obtained approx. 10 minutes after addition of 10 pM NiCh to 0.05 mg / mL UB-[Hise]2 in 30 mM Tris, pH 7.5, 25 °C. Figure 2B. Cryo-TEM image of 0.05 mg / mL UB-[Hise]2 following lOmin incubation in 30 mM Tris pH 7.5 at 25 °C with the addition of 10 mM ZnCh. Figure 2C. Cryo-TEM image of 3.8 mg / mL UB-[Hise]2 following overnight incubation in 30 mM Tris pH 7.5 at 19 °C with the addition of 100 mM ZnCh. Folded sheets / membranes are observed. Figure 2D. Cryo-TEM image of 3.8 mg / mL UB-[ Hiseh in in the absence of Ni2+or Zn2+with overnight incubation at 19 °C. FIG. 3A. Cryo-TEM imaging of protein sheets in vitreous ice obtained approx. 10 minutes after addition of 10 pM ZnCh to 0.05 mg / mL UB-[His6]2 in 30 mM Tris, pH 7.5, 25 °C.

[0079] FIGs. 3B-C. Magnification of dotted white rectangles (1 and 2) in 3A, respectively.

[0080] FIG. 4A. Cryo-TEM imaging of mono Hise-tagged Ubiquitin UB-[His6]i in vitreous ice obtained approx. 10 minutes after addition of 10 pM ZnCh to 0.05 mg / mE UB-[His6]i in 30 mM Tris, pH 7.5, 25 °C.

[0081] FIG. 4B. As in 3 A, but at higher magnification.

[0082] FIGs. 5A-D. Figure 5A-B. Cryo-TEM imaging of fibrous protein in vitreous ice obtained approx. 10 minutes after addition of 10 pM NiCP to 0.05 mg / mE Cas9-[Hise]2 in 30 mM Tris, pH 7.5, 25 °C. Figure 5C. Cryo-TEM image of 0.05 mg / mL Cas9-[Hise]2 following 10 min incubation in 30 mM Tris pH 7.5 at 25 °C with the addition of 10 mM ZnCh. Folded sheets / membranes are observed. Figure 5D. Cryo-TEM image of concentrated (3.8 mg / mL) Cas9-[His6]2 in the absence of Ni2+or Zn2+, incubated overnight in 30 mM Tris pH 7.5 at 19 °C. Organized supramolecular structures are not observed, rather disorganized aggregates.

[0083] FIGs. 6A-D. Far UV circular dichroism (CD) spectra of 0.05 mg / mL doubly His-tagged UB (Figures 6A-B) or Cas9 (Figures 6C-D) with 10 pM NiCh or ZnCh in DDW at 25 °C. Spectra measured in the absence of metal ions (— ) were used as control. Background has been subtracted.

[0084] FIGs. 7A-B. Gel electrophoresis of [UB-Hiseh and [Cas9-Hise]2: A. SDS-PAGE with reducing agent; B. Native-PAGE. Arrows indicate protein bands. Gels are Coomassie stained.

[0085] FIGs. 8A-C. Light microscopy images showing the impact of divalent cations on: Figure 8A. Doubly Hise-tagged ubiquitin (UB-[Hise]2); Figure 8B. Singly Hise-tagged ubiquitin (UB- [His6]2). In panels A and B, protein concentration 0.37 mM, 1 mM metal cations, following 1 hour incubation in the dark at 19°C in buffer (20 mM Tris pH 7.5). Figure 8C. Impact of water-soluble chelators on preformed UB-[Hise]2 aggregates. Control: UB-[Hise]2 as in panel A (+Zn2+); + (2.5 mM) EDTA; + (5 mM) Histidine; +(5 mM) imidazole. Images in the presence of EDTA, histidine or imidazole were taken 5 minutes after chelator addition. Scale bars represent 200 pm.

[0086] FIGs. 9A-B are cartoons illustrating how a supramolecular structure (fiber -Figure 9A; sheet - Figure 9B) of enzyme molecules generated according to embodiments of the invention may be used to detect a determinant. This embodiment illustrates how the sensitivity of a diagnostic assay such as ELISA may be intensified.

[0087] FIGs. 10A-B are cartoons illustrating how a supramolecular structure (fiber -Figure 10A; sheet - Figure 10B) of fluorescent molecules generated according to embodiments of the invention may be used to detect a determinant. This embodiment illustrates how the sensitivity of a diagnostic assay such as ELISA may be intensified. FIGs. 11A-B are cartoons illustrating how a supramolecular structure (fiber -Figure 10A; sheet - Figure 10B) composed of a both of fluorescent molecules and an antibody affinity agent, generated according to embodiments of the invention may be used to label an antibody. This embodiment illustrates how the sensitivity of a histological assay may be intensified.

[0088] FIG. 12 is a cartoon illustrating how double histidine-tagged proteins can be purified.

[0089] FIGs. 13A-C. Analysis of doubly and mono Hise-tagged mCherry. Figure 13A - 12% SDS- PAGE gel under reducing and non-reducing conditions. Lane 1: Molecular weight markers (Mw); Lanes 2-3: Doubly Hise-tagged mCherry (2.25 pgr) in the absence or presence of B- mercaptoethanol, respectively; lanes 4-5: mono Hise-tagged mCherry (1 pgr) in the absence or presence of B-mercaptoethanol, respectively; Figure 13B. As in A, but under native conditions. Lane 1: molecular weight markers; lane 2: mono Hise-tagged mCherry (1 pgr), lane 3: doubly Hise-tagged mCherry (2.25 pgr). Figure 13C. Illustration of postulated dimers of mCherry derivatives.

[0090] FIGs. 14A-D. Fluorescent imaging. Effect of indicated divalent cations (9 pM) on the mono Hise-tagged mCherry ((His) i -mCherry) (9 pM) after indicated times pointes at 8°C in DDW. Insets in B and D represent controls devoid of any cation. Bars in A-D represent 10 pm.

[0091] FIGs. 15A-D. Fluorescent imaging. Effect of indicated divalent cations (9 pM) on the doubly Hise-tagged mCherry ((His)2-mCherry) (9 pM) after indicated times pointes at 8 °C in DDW. Insets in B and D represent controls devoid of any cation. Bars in A-D represent 10 pm.

[0092] FIGs. 16A-B. Effect of indicated cations on the emission spectrum of (His)2-mCherry. Figure 16A. Initial emission spectrum of the doubly Hise-tagged mCherry (5 pM in DDW) in the absence (black line) or presence of 5 pM NiCh (green line) or 5 pM ZnCh (red line). Figure 16B. As in A, but after 3 hours of incubation in DDW at 25 °C.

[0093] FIGs. 17A-B. Figure 17A. CD analysis of 10 pM of (His)i-mCherry in the absence [black line] or presence of 10 pM Ni2+(green line) or Zn2+(red line) after indicated time points at 8°C in the dark. Figure 17B. CD analysis of 10 pM of (His)2-mCherry in the absence [black line] or presence of 10 pM Ni2+(green line) or Zn2+(red line) after indicated time points at 8°C in the dark.

[0094] FIGs. 18A-F: STEM analysis. A-E: Fibers and sheets generated in the presence of 9 pM of (His)2-mCherry and 3 pM of indicated cations (in DDW) after overnight incubation at 8°C in the dark. F. As in A-E, but in the absence of cations.

[0095] FIGs. 19A-D: STEM analysis. Figures 19A-C: Fibers and sheets generated in the presence of 9 pM of (His)i-mCherry and 3 pM of indicated cations (in DDW) after overnight incubation at 8°C in the dark. Figure 19D. As in A-C, but in the absence of cations. FIGs. 20A-B are photographs illustrating process reversibility. A. Fluorescent images showing the impact of indicated metal chelators after overnight incubation of 9 pM of (His)2- mCherry with 9 p M of Ni2+ at 8°C in the dark. Images in the presence of chelators were taken a few minutes after chelator addition. B. As in A, but in the presence of 9 pM of Zn2+.

[0096] FIG. 21 are photographs illustrating effect of pH on generation of supramolecular structures. Conjugation of 9 pM of (His)2-mCherry with 9 pM of Zn2+ at 8°C in the dark in the presence of 50 mM Na citrate at indicated pH values.

[0097] FIG. 22 is a photograph of a Coomassie-stained gel illustrating how generation of supramolecular structures aids in protein purification.

[0098] DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

[0099] The present invention, in some embodiments thereof, relates to methods of generating supramolecular structures of proteinaceous molecules using histidine tags and coordinating metal ions.

[0100] 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.

[0101] Whilst analyzing the properties of His-tagged proteins, the present inventors unexpectedly found that when the proteins contained more than two His tags, they generated supramolecular structures. This property was contingent upon the presence of metal ions.

[0102] This phenomenon was initially demonstrated for two different proteins ubiquitin (UB; MW < 10 kDa), Cas9 (>160 kDa), both expressed in, and purified from, E. coli BL21(DE3) cells. These experiments serve as a proof of principle for supramolecular polymerization of proteins via [metal: chelator] complexes interaction. Using cryo-TEM imaging, the formation of supramolecular protein fibers or sheets was observed, where the polymer building blocks are folded proteins, but only upon addition of divalent metal cations (Figures 2A-D, 3A-C, 4A-B and 5A-D). That protein secondary structure is preserved is validated by circular dichroism spectroscopy (Figures 6A-D). Ni2+gave rise to a fibrous morphology, while the addition of Zn2+primarily resulted in sheets / membranes. Thus, the present inventors were able to demonstrate biopolymerization of UB-[Hise]2 and Cas9-[His6]2 via [metakligand] chelation. Both nano-scale morphologies were obtained following relatively short incubation times at or near room temperature and at physiological pH, thereby opening the possibility of similar biopolymerization of labile proteins that do not tolerate acidic or basic conditions. Whilst further reducing the present invention to practice the present inventors successfully demonstrated the generation of supramolecular structures using the His-tagged mCherry protein (Figures 14A-D and 15A-D). The present inventors showed in this case that a single histidine tag was sufficient to promote generation of the supramolecular structures since the monomeric unit of the structure was a dimer composed of two mCherry molecules. Accordingly, each monomeric unit of the structure contained two His tags (as further explained in Figure 13C).

[0103] The dimeric state of the studied single and doubly His-tagged mCherry analogs allowed their assembly into filaments and sheets at pH ~7 only in the presence of equimolar (or less) amount of Zn2+or Ni2+. Preservation of the chromophore site in both analogs as well as their secondary structure provide direct evidence for the assembly mechanism mildness. The finding that native proteins clustered into gigantic ID or 2D assemblies (10-20 pm) under mild and specific conditions and that the process is fully reversible implies, that, the studied approach may introduce a novel type of supramolecular-biomaterials that were not generated thus far.

[0104] The present method of forming supramolecular structures has several advantages over other known methods for forming such structures. For example, the ability to direct proteins into filaments or sheets at equimolar (or less) Zn2+or Ni2+is expected to keep the protein of interest in its native conformation due to the low metal concentration used. In addition, the employed low metal concentration combined with the relatively tight affinity of the Hise-tag towards Zn2+(Kd = 0.047 pM) and Ni2+(Kd = 0.88 pM) implies that the concentration of free cations capable of binding non- specifically to the protein side chains and distort its functional state, is unlikely. Furthermore, the pH of the DDW used to prepare m-Cherry protein-fibers and sheets was very close to neutrality (z.e., pH 6.6-6.8). This suggests, that, other proteins may be manipulated similarly under mild conditions that would preserve their native conformation.

[0105] In addition, introduction of the His-tag is a very common approach for purification of recombinant proteins. Thus, insertion of two His-tags per protein monomer is not expected to represent a technical challenge. Moreover, the His-tag generally does not affect the native structure of proteins thus circumventing the need for its removal. Therefore, protein-filaments and proteinsheets composed of doubly His-tagged proteins are expected to preserve their biological roles.

[0106] The present inventors propose a myriad of uses of the proposed supramolecular structures.

[0107] For example, the present inventors further demonstrated that generation of supramolecular structures using histidine-tagged proteins allows for the purification of the protein by simple precipitation without the need of a column or resin (see Figures 12 and 22).

[0108] Other uses of the supramolecular structures include intensifying sensitivity of diagnostic assays such as ELISA (see Figures 9A-B, 10A-B), improving sensitivity of histological assays (Figures 11A-B). Each of these exemplary uses (as well as additional uses) is described more fully herein below.

[0109] Thus, according to an aspect of the invention, there is provided a method of generating a supramolecular structure comprising:

[0110] (a) contacting a divalent or trivalent metal cation with at least one protein-based molecule comprising at least two histidine tags under conditions that generate the supramolecular structure; and

[0111] (b) isolating the supramolecular structure.

[0112] As used herein the term “supramolecular structure” refers to an assembly of multiple proteinaceous repeating units that are non-covalently bound to form larger, functional structures. The structures typically comprise more than 10, 20, 30, 40, 50, 60, 70, 60, 90, 100, 200, 300, 400, 500 or more proteinaceous repeating units.

[0113] In one embodiment, the structure is a nanostructure - including nanofibers (wherein its length is in the nanoscale range; 1-100 nm) and nanosheets (wherein its surface area is in the nanoscale range: 1-100 nm2). Other examples of contemplated nanostructures include nanocages and nanogels.

[0114] In one embodiment, the structure is a microstructure - including microfibers (wherein its length is in the microscale range; 1-100 pm) and microsheets (wherein its surface area is in the microscale range: 1-100 pm2).

[0115] According to a particular embodiment, the structure is not a cyclic structure. Rather, the structure is extended (i.e., a fiber or a sheet).

[0116] The term “protein-based molecule” also referred to herein as a “proteinaceous repeating unit” refers to the repeating unit of the supramolecular structure. The protein-based molecule is comprised of amino acids which are bonded to one another.

[0117] The proteinaceous repeating unit may be a single protein monomer (in which the amino acids are covalently bonded to one-another), a protein dimer, a trimer or a larger repeating unit. The monomers of the dimeric or trimeric repeating unit are typically non-covalently bound to each other, but rather are bound by ionic, hydrogen, Van der Waals (VW) forces, [71:71] stacking. The proteins of the dimeric or trimeric repeating unit are not coordinated to one another by metal ions.

[0118] The present inventors contemplate that the proteinaceous repeating unit of the supramolecular structure is composed of a single protein or multiple proteins (e.g., 2, 3, 4, 5 or more) of different identities.

[0119] The proteins typically comprise the wild-type (non-mutated) sequence of the protein, although it will be appreciated, mutated proteins are also contemplated. It will be appreciated that the term “mutated protein” does not refer to a protein to which a tag has been added.

[0120] In one embodiment, the protein retains its function in the supramolecular structure.

[0121] The proteins may be alpha-helical proteins, beta barrel shaped proteins or any other 3D structure.

[0122] In one embodiment, the protein which is comprised in the supramolecular structure has no natural tendency to oligomerize. Thus, for example the protein of the supramolecular structure is not an amyloid or amyloid-lik protein.

[0123] The protein-based molecules in the structure are not typically limited by virtue of their molecular weight. Typically, the protein-based molecule is at least 5 amino acids or at least 10 amino acids in length. Exemplary molecular weights of protein-based molecules are between 1 KDa and 500 KDa).

[0124] In one embodiment, at least one of the molecular-based proteins is a water soluble protein.

[0125] In one embodiment, none of the molecular-based proteins are amphiphilic proteins.

[0126] In another embodiment, at least one of the molecular-based proteins is a detectable protein (e.g. fluorescent, phosphorescent, enzyme-based detectable protein).

[0127] Methods of detecting the detectable moiety are known in the art and are dependent upon the type of detectable moiety used in the assay. In one embodiment, the detectable moiety is a fluorescent moiety and the method of detecting is by fluorescent imaging.

[0128] Exemplary protein-based fluorophores used in the supramolecular structures of the present invention may include, but are not limited to Green fluorescent protein (GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), red fluorescent protein (RFP), mCherry, mVenus and mCerulean3.

[0129] Enzyme based detectable proteins used in the supramolecular structures of the present invention, include, but are not limited to horseradish peroxidase, alkaline phosphatase, B- galactosidase, Luciferase, glucose oxidase, caspase enzyme, urease and alcohol dehydrogenase.

[0130] In another embodiment, at least one of the protein-based molecules of the supramolecular structures binds specifically (e.g., with a Kd between 1-10 nM or between 1-5 nM) to the Fc region of an antibody. Such molecules include Protein A (which binds to the Fc region of IgG), Protein G (which binds to the Fc region of IgG) and Protein L which binds to Light chains. Shorter fragments of these proteins are also contemplated - for example The E domain, D domain, A domain, B domain and C domain of Protein A. The Z domain (an engineered stable version of domain B) is also contemplated.

[0131] An exemplary amino acid sequence of the Z domain is set forth in SEQ ID NO: 9. An exemplary sequence of a double histidine-tagged Z sequence is provided in SEQ ID NO: 10.

[0132] It will be appreciated that the histidine-tagged Z sequence may comprise more than one repeat of the Z sequence - for example two, three or more consecutive Z sequences. Thus, for example the Z sequence may comprise the following sequence - SEQ ID NO:

[0133] 11 or 12.

[0134] In another embodiment, the supramolecular structure comprises a combination of detectable protein-based molecule and a protein-based molecule that binds to the Fc region of an antibody - (see Figures 9A-B, 10A-B and 11A-B for example).

[0135] Other exemplary protein-based molecules which may be incorporated in the supramolecular structures include but are not limited to receptors, enzymes, membrane proteins, antibodies, antibody fragments, aptamers and structural proteins.

[0136] In one embodiment, the protein is capable of conducting an electrical current. Examples of such proteins include, but are not limited to cytochromes, ferrodoxins, plastocyanin and azurin. The present inventors contemplate using fibers of such proteins as molecular wires.

[0137] In another embodiment, the protein-based molecule is an antigenic peptide or protein. The antigenic peptide may be one that is capable of being presented on the surface of a cell bound to an Major Histocompatibility Complex (MHC) molecule, or of being recognized by a T receptor.

[0138] The protein-based molecules comprised in the structures comprise at least two histidine tags.

[0139] As used herein, the term “histidine tag” refers to a repeat sequence of histidines that is not part of the amino acid sequence of the wild-type protein. Each histidine tag may comprise between 2-10 or between 3-8 consecutive histidines. In one embodiment, the histidine tag comprises 6 consecutive histidines. Typically, the Histidine tag is included in the amino acid sequence of the protein such that it does not interfere with the structure and / or function of the protein. Typically, the histidine tags are at least 5, 10, 20, 30 or more amino acids apart from each on the protein molecule.

[0140] In one embodiment, one of the histidine tags is at the N-terminus of the protein molecule and the other of the histidine tags is at the C-terminus of the protein-based molecule. In the case where the protein molecule is a dimer, each monomer of the protein molecule may be labeled with a histidine on the C-terminus or the N-terminus.

[0141] Generation of histidine tagged proteins can be carried out using known molecular biology techniques. To produce a histidine tagged protein using recombinant technology, an isolated polynucleotide comprising a nucleic acid sequence encoding such a polypeptide may be used.

[0142] The term "nucleic acid sequence" refers to a deoxyribonucleic acid sequence composed of naturally-occurring bases, sugars and covalent intemucleoside linkages (e.g., backbone) as well as oligonucleotides having non-naturally-occurring portions which function similarly to respective naturally-occurring portions. Such modifications are enabled by the present invention provided that recombinant expression is still allowed.

[0143] A nucleic acid sequence of the histidine-tagged protein according to this aspect of the present invention can be a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and / or a composite polynucleotide sequences (e.g., a combination of the above).

[0144] As used herein the phrase "complementary polynucleotide sequence" refers to a sequence, which results from reverse transcription of messenger RNA using a reverse transcriptase or any other RNA dependent DNA polymerase. Such a sequence can be subsequently amplified in vivo or in vitro using a DNA dependent DNA polymerase.

[0145] As used herein the phrase "genomic polynucleotide sequence" refers to a sequence derived (isolated) from a chromosome and thus it represents a contiguous portion of a chromosome.

[0146] As used herein the phrase "composite polynucleotide sequence" refers to a sequence, which is at least partially complementary and at least partially genomic. A composite sequence can include some exonal sequences required to encode the polypeptide of the present invention, as well as some intronic sequences interposing therebetween. The intronic sequences can be of any source, including of other genes, and typically will include conserved splicing signal sequences. Such intronic sequences may further include cis acting expression regulatory elements.

[0147] Linking of a polynucleotide sequence which encodes histidine tag and a polynucleotide sequence that encodes a protein of interest may be effected using standard molecular biology techniques including the use of PCR, ligation enzymes and restriction enzymes. In one embodiment, the 5’ end of the histidine tag is ligated to the 3’ end of the gene encoding the protein of interest such that a histidine-tagged protein is generated with a histidine tag at the N terminus and the polypeptide of interest at the C terminus. In another embodiment, the 3’ end of the histidine tag is ligated to the 5’ end of the gene encoding the protein of interest such that a histidine- tagged protein is generated with a histidine tag at the C terminus and the polypeptide of interest at the N terminus. In still another embodiment, the histidine tag is added to both the N and C terminus of the protein of interest. In one embodiment, the histidine tag is added to the N and / or C terminus with a linker peptide in between. The linker peptide typically is a flexible linker. Peptide linkers may be entirely artificial (e.g., comprising 2 to 20 amino acid residues independently selected from the group consisting of glycine, serine, asparagine, threonine and alanine) or adopted from naturally occurring proteins. An exemplary linker peptide is SEQ ID NO: 13 (SAGSAGSAG) or SEQ ID NO: 14 (SRADPKKKRKVAAALE).

[0148] In order to generate the histidine-tagged proteins of the present invention using recombinant techniques, the polynucleotides encoding same are ligated into nucleic acid expression vectors, such that the polynucleotide sequence is under the transcriptional control of a cis-regulatory sequence (e.g., promoter sequence).

[0149] A variety of prokaryotic or eukaryotic cells can be used as host-expression systems to express the polypeptides of the present invention. These include, but are not limited to, microorganisms, such as bacteria transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing the polypeptide coding sequence; yeast transformed with recombinant yeast expression vectors containing the polypeptide coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors, such as Ti plasmid, containing the polypeptide coding sequence.

[0150] Exemplary bacterial cells that may be used to express the histidine-tagged protein are E. coli, such as an E. coli protein deficient strain, e.g. E. coli BL21 or Rosetta gami-2.

[0151] Exemplary yeast cells that may be used to express the histidine-tagged protein are K. lactis or S. cerevisiae.

[0152] Constitutive promoters suitable for use with this embodiment of the present invention include sequences which are functional (i.e., capable of directing transcription) under most environmental conditions and most types of cells such as the cytomegalovirus (CMV) and Rous sarcoma virus (RSV).

[0153] Inducible promoters suitable for use with this embodiment of the present invention include for example the tetracycline-inducible promoter (Srour, M.A., et al., 2003. Thromb. Haemost. 90: 398-405) or the lac operator. In the latter case, gene expression is induced using Isopropyl -D-1- thiogalactopyranoside (IPTG) at a concentration between 0.1 mM - ImM at a temperature between 20-30 °C (for example 25 °C).

[0154] The expression vector according to this embodiment of the present invention may include additional sequences which render this vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors). Typical cloning vectors contain transcription and translation initiation sequences (e.g., promoters, enhances) and transcription and translation terminators (e.g., poly adenylation signals). Eukaryotic promoters typically contain two types of recognition sequences, the TATA box and upstream promoter elements. The TATA box, located 25-30 base pairs upstream of the transcription initiation site, is thought to be involved in directing RNA polymerase to begin RNA synthesis. The other upstream promoter elements determine the rate at which transcription is initiated.

[0155] Enhancer elements can stimulate transcription up to 1,000 fold from linked homologous or heterologous promoters. Enhancers are active when placed downstream or upstream from the transcription initiation site. Many enhancer elements derived from viruses have a broad host range and are active in a variety of tissues. For example, the SV40 early gene enhancer is suitable for many cell types. Other enhancer / promoter combinations that are suitable for the present invention include those derived from polyoma virus, human or murine cytomegalovirus (CMV), the long term repeat from various retroviruses such as murine leukemia virus, murine or Rous sarcoma virus and HIV. See, Enhancers and Eukaryotic Expression, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 1983, which is incorporated herein by reference.

[0156] Poly adenylation sequences can also be added to the expression vector in order to increase the translation efficiency of a polypeptide expressed from the expression vector of the present invention. Two distinct sequence elements are required for accurate and efficient polyadenylation: GU or U rich sequences located downstream from the polyadenylation site and a highly conserved sequence of six nucleotides, AAUAAA, located 11-30 nucleotides upstream. Termination and polyadenylation signals that are suitable for the present invention include those derived from SV40.

[0157] In addition to the elements already described, the expression vector of the present invention may typically contain other specialized elements intended to increase the level of expression of cloned nucleic acids or to facilitate the identification of cells that carry the recombinant DNA. For example, a number of animal viruses contain DNA sequences that promote the extra chromosomal replication of the viral genome in permissive cell types. Plasmids bearing these viral replicons are replicated episomally as long as the appropriate factors are provided by genes either carried on the plasmid or with the genome of the host cell.

[0158] The vector may or may not include a eukaryotic replicon. If a eukaryotic replicon is present, then the vector is amplifiable in eukaryotic cells using the appropriate selectable marker. If the vector does not comprise a eukaryotic replicon, no episomal amplification is possible. Instead, the recombinant DNA integrates into the genome of the engineered cell, where the promoter directs expression of the desired nucleic acid. Examples of bacterial expression vectors suitable for the present invention include but are not limited to pET21b+, pBR322 or pET28+.

[0159] Examples for mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(+ / -), pGL3, pZeoSV2(+ / -), pSecTag2, pDisplay, pEF / myc / cyto, pCMV / myc / cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMTl, pNMT41, pNMT81, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.

[0160] Expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses can also be used by the present invention. SV40 vectors include pSVT7 and pMT2. Vectors derived from bovine papilloma virus include pBV-lMTHA, and vectors derived from Epstein Bar virus include pHEBO, and p2O5. Other exemplary vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.

[0161] Various methods can be used to introduce the expression vector of the present invention into cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.

[0162] Transformed cells are cultured under effective conditions, which allow for the expression of high amounts of recombinant polypeptide. Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production. An effective medium refers to any medium in which a cell is cultured to produce the recombinant polypeptide of the present invention. Such a medium typically includes an aqueous solution having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins.

[0163] Cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes and petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Such culturing conditions are within the expertise of one of ordinary skill in the art.

[0164] Depending on the vector and host system used for production, resultant polypeptides of the present invention may either remain within the recombinant cell, secreted into the fermentation medium, secreted into a space between two cellular membranes, such as the periplasmic space in E. coli; or retained on the outer surface of a cell or viral membrane.

[0165] Following a predetermined time in culture, recovery of the recombinant histidine-tagged protein is effected.

[0166] If the histidine-tagged protein is expressed in the cell, the cell membrane is preferably disrupted so as to release the histidine-tagged protein (e.g. by sonication, detergent or freeze-thaw).

[0167] The histidine-tagged protein may be purified using methods known in the art including for example heat denaturation, salt induced precipitation, mixed mode chromatography, cation exchange chromatography and anion exchange chromatography.

[0168] According to a particular embodiment, the histidine-tagged proteins are purified using a resin which binds metal ions (Zinc (Zn2+), Nickel (Ni2+), iron (Fe2+or Fe3+), or cobalt (Co2+). The resin is typically washed and the protein is then eluted using imidazole (e.g. 250-500 mM) to compete with the His-tag.

[0169] As mentioned, once the histidine tagged protein molecules are obtained, supramolecular structures may be generated by contacting them (in solution) with divalent or trivalent ions.

[0170] Contacting is effected under conditions that allow generation of the supramolecular structures and further that allow the protein molecules of the structure to retain functionality. Contacting should be effected for sufficient time (e.g. at least 10 minutes), at an appropriate temperature (e,g., between 4 °C - 37 °C), pH (e.g., between pH 5-8) etc. The molecular ratio of [histidine tagged proteins : coordinating metal ions] may be between [1:10] - [10:1].

[0171] In one embodiment, the divalent or trivalent ions of the structure coordinate non-covalent binding of 2-6 proteinaceous repeating units.

[0172] In the case, wherein the divalent or trivalent ions of the structure coordinate non-covalent binding of 2 proteinaceous repeating units, a fiber is formed.

[0173] In the case, wherein the divalent or trivalent ions of the structure coordinate non-covalent binding of 4 proteinaceous repeating units, a sheet is formed.

[0174] Typically, the metal ions are the only means for connecting the protein-based molecules to one another. Thus, for example the supramolecular structure does not rely on the use of amphiphilic peptides to connect between one protein-based molecule to another. Examples of divalent or trivalent ions that can be used to form the supramolecular structures include Zinc (Zn2+), Nickel (Ni2+), iron (Fe2+or Fe3+), or cobalt (Co2+).

[0175] According to a particular embodiment, the divalent ion is Zinc (Zn2+) or Nickel (Ni2+).

[0176] The molecular weight of the supramolecular structure is typically greater than 50 KDa, greater than 100 KDa or even greater than 200 KDa.

[0177] Once generated, the supramolecular structures can be isolated. Since the structures are so large, an easy method of isolation includes precipitation. Following isolation, typically the structure is devoid of smaller structures (i.e., those that contain only up to 2, 3, or 4 protein-based molecules).

[0178] Thus, according to another aspect of the invention, there is provided a composition comprising a purified supramolecular structure which comprises at least two repeating units, each repeating unit being represented by the formula P-D-P-D, wherein

[0179] P is a protein-based molecule comprising at least two histidine tags (as described herein above); and

[0180] D is a divalent or trivalent ion, wherein each protein-based molecule in the structure is non-covalently bound to at least one other protein-based molecule via the divalent or trivalent ion.

[0181] The divalent or trivalent ion of the structure coordinates non-covalent binding of between 2-6 protein-based molecules.

[0182] In one embodiment, the divalent ion of the structure coordinates non-covalent binding of 2 protein-based molecules and a fiber is formed.

[0183] In one embodiment, the divalent ion of the structure coordinates non-covalent binding of 4 protein-based molecules and a sheet is formed.

[0184] According to this aspect, the structure comprises at least four histidine-tagged proteinbased molecules each connected to at least one other protein based molecule via a divalent or trivalent ion.

[0185] The structure may comprise at least one, two, three, four, five or more different identities of protein-based molecules.

[0186] Further, the structure may comprise more than one type of divalent ion or trivalent ion.

[0187] The size, 3D structure, protein identity and other characteristics have been described herein above.

[0188] The purified supramolecular structure is typically devoid of (or only has small quantities of) smaller structure which do not have at least four protein based molecules. In one embodiment, the purified supramolecular structure is typically devoid of (or only has small quantities of) structures represented by the formula P-D-P.

[0189] The supramolecular structures of any of the embodiments of the invention may be attached (i.e. immobilized) to a solid surface using methods known in the art. For example, the supramolecular structures may comprise an immobilizing moiety that interacts with a corresponding moiety on the solid surface. Examples of such immobilizing moieties include, but are not limited to (a) ligand: receptor (b) antigen: antibody (c) biomarker: antibody (d) biomarker: fragment of an antibody (e) target biomolecule: aptamer (f) target biomolecule: antibody fragment (g) target biomolecule:Fc-fusion protein

[0190] Thus, according to aspects of the present invention, there is provided an article of manufacture comprising a solid surface attached to the supramolecular structures described herein.

[0191] The article of manufacture may be fabricated from materials including, but not limited to silicon, nitrocellulose, cellulose acetate, nylon, polyvniylidene Fluoride (PVDF), polystyrene, polyacrylamide, chitosan, agarose and glass.

[0192] In one embodiment, the article of manufacture is a medical device.

[0193] For example, the medical device may be a stent. The supramolecular structure may comprise an anti-thrombic or endothelial mimicking protein (e.g. fibronectin, laminin or VEGF), which can promote endothelialization and reduce immune rejection.

[0194] In another embodiment, the medical device is a hemodialysis catheter or blood filter. The supramolecular structure may comprise albumin or heparin-binding proteins to reduce protein adsorption and platelet activation.

[0195] In another embodiment, the medical device is neural implant or electrode. The supramolecular structure may comprise neural adhesion proteins such as laminin or NCAM fragments to enhance integration into neural tissue.

[0196] In another embodiment, the medical device is an injectable of implantable drug delivery system. The supramolecular structure may comprise proteins that act as release regulators or biological recognition sites.

[0197] In another embodiment, the medical device is a contact lens or ocular implant. The supramolecular structure may comprise mucin like proteins or growth factors to mimic natural tear film or promote corneal healing.

[0198] In another embodiment, the article of manufacture is a detection device - i.e. one which allows detection of a detectable moiety. Examples of such include biosensors, lateral flow assays, microarrays, protein chips, lab on a chip devices, electrochemical or optical probes, point of care blood analyzers etc. In another embodiment, the article of manufacture is a microplate which comprises wells. The well may be the size of a well of a standard 384- well plate or 96-well plate or a 24- well plate or 12- well plate or 6- well plate, as known in the art. In another embodiment, the article of manufacture is a tube, or a flask. In another embodiment, the article of manufacture is a cell-perfusion system.

[0199] The term “cell perfusion system” refers to a system which allows biological cells to be cultured, while continuously exchanging culture medium. Fresh medium replenishes nutrients and carbon sources, while cellular waste and medium depleted of nutrients are removed.

[0200] As mentioned, the supramolecular structures described herein may serve a myriad of uses.

[0201] In one embodiment, the supramolecular structures serve to enhance purification of a protein of interest.

[0202] The protein of interest may be comprised in the supramolecular structure i.e. the protein of interest is “P” of the above described formula “P-D-P-D”. This aspect is described in Figure 12 of the application. Upon formation of a supramolecular structure, a precipitate is generated. The supernatant is removed and optionally washed. The His tagged protein molecules of the supramolecular structure may be released by adding a chelator such as EDTA, imidazole or histidine. This method was successfully carried out using mCherry as the protein of interest (see Figure 20).

[0203] The supramolecular structures may also serve to enhance purification of other biomolecules (which are not incorporated in the supramolecular structures).

[0204] Thus, according to one aspect of the invention there is provided a method of isolating a biomolecule from a solution comprising:

[0205] (a) contacting the biomolecule with a composition under conditions that allows binding of the biomolecule to the biomolecule-binding protein; and

[0206] (b) removing the supramolecular structure from the solution wherein the composition comprises a purified supramolecular structure which comprises at least two repeating units, each repeating unit being represented by the formula P-D-P-D, wherein P is a biomolecule binding -protein comprising at least two histidine tags; and D is a divalent or trivalent ion, wherein each biomolecule binding-protein in the structure is non-covalently bound to at least one other biomolecule binding-protein via the divalent or trivalent ion.

[0207] Examples of biomolecules which can be isolated include but are not limited to proteins, nucleic acids, lipids, sugars and small molecules. In one embodiment, the biomolecule is a protein and the biomolecule binding-protein is an antibody or antibody fragment capable of binding the protein.

[0208] In another embodiment, the biomolecule is an antibody and the biomolecule bindingprotein is a protein binds to an FC region of an antibody (for example, Protein G or Protein A or fragments thereof, as further described herein above).

[0209] Upon binding of the supramolecular structure to the biomolecule, a precipitate comprising the biomolecule is generated. The supernatant is removed and optionally washed. The His tagged protein molecules of the supramolecular structure (which are bound to the biomarker) may be released by adding a chelator such as EDTA, imidazole or histidine.

[0210] The supramolecular structures described herein may also serve as diagnostic agents or detection agents generating a signal when a biomarker is detected.

[0211] Thus, according to another aspect of the present invention there is provided a biomolecule affinity agent comprising a supramolecular structure which comprises at least two repeating units, each repeating unit being represented by the formula P-D-P-D, wherein

[0212] P is a detectable protein-based molecule comprising at least two histidine tags; and

[0213] D is a divalent or trivalent ion, wherein at least one detectable protein-based molecule in the structure is non-covalently bound to a second detectable protein-based molecule via the divalent or trivalent ion, wherein the supramolecular structure further comprises a protein that binds directly or indirectly to the biomolecule, the protein being non-covalently bound to the detectable proteinbased molecule via the divalent or trivalent ion.

[0214] Biomolecules that may be detected include but are not limited to proteins, nucleic acids, carbohydrates, sugars.

[0215] In one embodiment, the biomolecule is an isolated biomolecule. In another embodiment, the biomolecule is a comprised on the outer surface of (or in) a cell - see for example Figures 11A-B.

[0216] Detectable protein-based molecules which can be incorporated in the biomolecule affinity agents have been described herein above and include for example fluorescent proteins (see for example, Figures 10A-B), enzyme-based proteins (see for example, Figures 9A-B) etc.

[0217] The supramolecular structure of the biomolecule affinity agent may be a fiber, a sheet or any other structure as described herein above.

[0218] Exemplary proteins that bind (directly, or indirectly) to a protein biomolecule include antibodies, proteins that bind to FC regions of antibodies, as further described herein above. It will be appreciated that in order to detect a protein biomolecule using a protein (or protein fragment) that binds to an FC region of an antibody in the supramolecular structures, the biomolecule is typically initially contacted with an antibody that binds directly and specifically to the biomolecule. The biomolecule affinity agents are then contacted with the antibody-tagged biomolecules such that the FC-binding protein binds to the antibody. The biomolecule affinity agents serve to concentrate the number of detectable protein-based molecules providing a strong detection signal and enabling detection of very small amounts of biomolecule.

[0219] According to this aspect both the biomolecule binding protein and the detectable proteinbased molecule comprises at least two histidine tags, as further described herein above.

[0220] The ratio of biomolecule binding protein: detectable protein based molecule may be any ratio that allows for efficient incorporation of both protein in the supramolecular structure. In one embodiment, the ratio of biomolecule binding protein: detectable protein based molecule is between 100:1 - 1:100. In particular embodiments the ratio of biomolecule binding protein detectable protein based molecule may be between 10:1 - 1:10. In still further embodiments, the ratio of biomolecule binding protein detectable protein based molecule is at least between 3:1 - 1:3.

[0221] The biomolecule affinity agent may be attached to a solid surface using methods known in the art, such as using methods described herein above.

[0222] The supramolecular structures may be used to concentrate a component of a chemical reaction, e.g. a catalytic reaction.

[0223] Thus, according to still another aspect of the present invention, there is provided a method of performing a catalytic (e.g. an enzymatic) reaction comprising contacting a composition with starting components of the cataltyic reaction under conditions that allow the catalyst (e.g. enzyme) to catalyze the enzymatic reaction, thereby performing the enzymatic reaction; wherein the composition comprises a supramolecular structure which comprises at least two repeating units, each repeating unit being represented by the formula P-D-P-D, wherein

[0224] P is a catalyst (e.g. enzyme) molecule comprising at least two histidine tags; and

[0225] D is a divalent or trivalent ion, wherein each catalyst-molecule in the structure is non-covalently bound to at least one other catalyst-molecule via said divalent or trivalent ion.

[0226] The term “enzyme” refers to a protein that speeds up chemical reactions that is not consumed in the process. In one embodiment, at least 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 % 90 % or all of the enzyme molecules in the supramolecular structure are orientated such that the catalytic site thereof has access to the enzyme substrates.

[0227] Exemplary enzymes include alkaline phosphatase or horseradish peroxidase.

[0228] The supramolecular structures described herein may be used to enhance production of antibodies. Thus, according to still another aspect there is provided a method of generating an antibody in a subject comprising administering to the subject the supramolecular structures described herein, thereby generating the antibody.

[0229] It will be appreciated that the protein-based molecule of the supramolecular structure is the protein to which the antibody targets.

[0230] The supramolecular structure acts as a carrier protein to enhance the immunogenicity of the protein. Thus, the supramolecular structure acts as a similar way to KLH (Keyhole Limpet Hemocyanin) or Bovine serum albumin (BSA).

[0231] Use of the supramolecular structures of the invention is particular relevant when the protein-based molecules are too small to elicit a strong immunogenic response - e.g. less than 10 KDa or less than 8 KDa. The resulting immunogenic complex can then be injected into suitable mammalian subjects such as mice, rabbits, and the like. Suitable protocols involve repeated injection of the immunogen in the presence of adjuvants according to a schedule which boosts production of antibodies in the serum. The titers of the immune serum can readily be measured using immunoassay procedures which are well known in the art.

[0232] The antisera obtained can be used directly or monoclonal antibodies may be obtained as described hereinabove.

[0233] In one embodiment, the supramolecular structures can be used as a vaccine and administered to a human subject. The supramolecular structures act as an adjuvant enhancing the immune reaction to the protein incorporated therein.

[0234] As used herein the term “about” refers to ± 10 %

[0235] The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

[0236] The term “consisting of’ means “including and limited to”.

[0237] 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.

[0238] 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.

[0239] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0240] 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.

[0241] 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.

[0242] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. EXAMPLES

[0243] 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.

[0244] 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. I., 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

[0245] SUPRAMOLECULAR STRUCTURES OF HIS-TAGGED UBIQUTIN AND CAS9

[0246] MATERIALS AND METHODS

[0247] Materials

[0248] NaCl - Sodium chloride, MgCh - Magnesium chloride, Zinc chloride (ZnCh), Nickel chloride (NiCh), Tris, Isopropyl beta-D-1 -thiogalactopyranoside (IPTG), Imidazole, phenylmethylsulphonyl fluoride (PMSF), Ethylenediaminetetraacetic acid (EDTA) were all purchased from Sigma-Aldrich, Israel; pET28a (Novagen - Merck, Germany); BL21(DE3) (Thermo Fisher Scientific, USA); lysozyme (Thermo Fisher Scientific, USA); DNase (Invitrogen - Israel).

[0249] Methods

[0250] Cloning expression and purification of ubiquitin (UB)-[Hise]2 pET28-human ubiquitin plasmid without a tag was used as a template for construction of the double hexa-His tag plasmid. Addition of the double hexa-His tag was performed by Transfer- PCR (TPCR) using the relevant primers (Table 1). Sequence analysis was performed to ensure the integrity of the UB-[Hise]2 gene. The final construct pET28-UB-[Hise]2 contains hexa-His-tag and linker at the N-terminal (MGSSHHHHHHSAGSAGSAG - SEQ ID NO: 1) and a linker and hexa- His-tag at the C-terminus (SAGSAGSAGHHHHHH - SEQ ID NO: 2). pET28-UB-[His6]2was expressed in LB media in BL21(DE3) cells using Kanamycin (30mg / ml) as a selection. A 5 L culture was induced at ODeoo 0.6-0.8, with 200 pM isopropyl beta-D-1 -thiogalactopyranoside (IPTG) and grown at 15 °C overnight. The culture was harvested and lysed by a cooled cell disrupter (Constant Systems) in lysis buffer (50 mM Tris pH=8, 0.5 M NaCl, 20 mM imidazole) containing 200 KU / 100 mL lysozyme, 20 pg / mL DNase, 1 mM MgCh, ImM phenylmethylsulphonyl fluoride (PMSF) and protease inhibitor cocktail. Following clarification of the supernatant by centrifugation, the lysate was applied to a HisTrap-FF_5 mL column (GE Healthcare) and eluted with binding buffer containing 0.5 M imidazole. The eluted Ub- [ H i se] 2 was injected into a size exclusion (SEC) column (HiLoad_16 / 60_Superdex75 prep-grade, GE Healthcare) equilibrated with 50 mM Tris pH=8, 200 mM NaCl, 1 mM EDTA. Pure UB-[Hise]2 migrated as a single peak at 85 mL, was pooled and flash frozen in aliquots using liquid nitrogen and stored at -80 °C. Table 1

[0251] Cloning expression and purification of Cas9 with double hexa-His tag: pET28-spCas9 (addgene # 47327) with a single C-terminal hexa-His-tag was used as a template for construction of the double hexa-His tag construct. Addition of the N-terminal Hexa- His-tag was performed by inverse-PCR using His_Cas9_F and His_Cas9_R primers (Table 1). The linear PCR product was ligated using the KLD enzyme mix (New England Biolabs, M05544). Sequence analysis was performed to ensure the integrity of the spCas9-[Hise]2 gene. The final construct pET28-spCas9-[His6]2 contains hexa-His-tag and linker at the N-terminal (MGSSHHHHHHSAGSAGSAG - SEQ ID NO: 1) and a hexa-His-tag and linker at the C- terminus (SRADPKKKRKVAAALEHHHHHH - SEQ ID NO: 3). Expression of pET28-spCas9- [His6]2was performed in LB media in BL21(DE3) cells using Kanamycin (30 mg / ml) as a selection. A 5 L culture was induced at ODeoo 0.6-0.8 with 200 pM IPTG and grown at 15 °C overnight. The culture was harvested and lysed by a cooled cell disrupter (Constant Systems) in lysis buffer (50 mM Tris pH=8, 0.5 M NaCl, 20 mM imidazole) containing 200 KU / 100 mL lysozyme, 20 pg / mL DNase, 1 mM MgCh, 1 mM phenylmethylsulphonyl fluoride (PMSF) and protease inhibitor cocktail. Following clarification of the supernatant by centrifugation, the lysate was applied to a HisTrap-FF_5 mL column (GE Healthcare) and eluted with binding buffer containing 0.5 M imidazole. The eluted Cas9-[Hise]2 was injected into a size exclusion (SEC) column (HiLoad_16 / 60_Superdex200 prep-grade, Cytivia) equilibrated with 20 mM Tris pH=8, 200 mM KC1, 10 mM MgCh. Pure Cas9-[Hise]2, migrating as a single peak at 66 mL, was pooled and flash frozen in aliquots using liquid nitrogen and stored at -80 °C. Following thawing, both UB and Cas9 were dialyzed against 30-50 mM Tris, pH 7-7.5 at 8 °C for 24 hours prior to all subsequent measurements. Cryo-TEM imaging of UB-[Hise]2and Cas9-[Hise]2

[0252] Cryo-TEM samples were prepared in a Leica GP2 plunger (Leica Microsystems, Germany) on lacy carbon grids, pretreated with a 1-min glow discharge in PELCO EasiGlow appliance (Ted Pella, Inc., USA), to render the carbon film more hydrophilic. In the absence of added divalent cations, 5 pL of 3.8 mg / mL UB-[His6]2 or Cas9-[His6]2 in 50 mM NaHCOs (pH 6) was applied to the carbon grid in the plunger chamber set to 90% relative humidity, 25 °C. To detect metal / ligand conjugation, 2 pL of 1 mM divalent cations (ZnCh or NiCh) in 196 pL of 30 mM Tris buffer, pH 7.5, 25 °C were added to 2 pL of 3.8 mg / mL UB-[Hise]2 or Cas9-[His6]2 in the same buffer, but where the latter contained 100 mM NaCl as well. 4 pL of the mixture was then applied to the grid. Grids were blotted for 5 seconds using No. 1 Whatman paper and then were plunge-frozen in liquid ethane, cooled by liquid nitrogen to ensure specimen vitrification (fast-freezing). This largely prevents ice crystal formation. Vitrified specimens were stored in liquid nitrogen until viewing in the microscope. For imaging, cryo- specimens were loaded under controlled conditions into a Gatan 626 cryo-holder and kept in the TEM at -180 °C during the entire procedure. Imaging was performed with a FEG-equipped Talos 200 C TEM (Thermo Fisher Scientific, USA), operated at 200 kV, using the low-dose software of the TEM. Images were recorded by a Falcon III direct- imaging camera using the TIA software package.

[0253] Far UV circular dichroism (CD) spectroscopy

[0254] Samples were dissolved at 0.05 mg / mL in double distilled water (DDW) and subjected to CD analysis using a Chirascan CD spectrometer (Applied Photophysics). CD spectra report ellipticity (0), proportional to the difference in absorbance of left and right circularly polarized light [9 = 3300° (AL~ AR)] as a function of wavelength. A quartz cell of path length 0.1 cm was used for the measurements. The CD spectra were recorded with 2 nm bandwidth resolution in 1 nm steps at 25 °C. CD spectra were corrected for baseline distortion by subtracting a reference spectrum of the corresponding solvent.

[0255] SDS-PAGE electrophoresis

[0256] 5 pL of 3.8 mg / mL of UB-[Hise]2 or 10 pL of 0.38 mg / mL of Cas9-[Hise]2 were loaded onto a 10% polyacrylamide gel, in the presence of SDS and beta mercaptoethanol, prepared according to the protocol of Laemmli [Nature, 1970. 227(5259): p. 680-5]. The low molecular weight of UB necessitated increasing the amount of protein run on the gel 10-fold relative to Cas9 in order that the Coomassie staining would provide sufficient contrast.

[0257] Native polyacrylamide gel electrophoresis (Native-PAGE)

[0258] 5 pL of 3.8 mg / mL of UB-[Hise]2 or 10 pL of 0.38 mg / mL of Cas9-[Hise]2 were loaded onto a 7.5% native polyacrylamide gel, in the absence of SDS or any reducing agent, prepared according to the protocol of Trudel and Asselin [Anal Biochem, 1994. 221(1): p. 214-216]. The low molecular weight of UB necessitated increasing the amount of protein run on the gel 10-fold relative to Cas9 in order that the Coomassie staining would provide sufficient contrast.

[0259] Light microscopy

[0260] Hanging drops containing 0.37 mM doubly or singly Hise-tagged ubiquitin (UB-[His6]2 or UB-[His6]i) were incubated with 1 mM divalent cations in 20 mM Tris pH 7.5 in the dark for 1 hour at 19 °C. The effect of 2.5 mM EDTA, 5 mM histidine or 5 mM imidazole was evaluated 5 minutes after chelator addition using the Olympus CX-40 light microscope equipped with an Olympus U-TV1X-2 digital camera.

[0261] RESULTS

[0262] Macromolecular UB-[Hise]2 polymerization promoted by ligand-divalent cation interactions

[0263] Using cryo-TEM imaging, it was possible to observe the formation of ubiquitin (UB) fibers following 10 minutes room temperature incubation of UB-[Hise]2 (0.05 mg / mL, ~ 6 pM) with 10 pM Ni2+ions in Tris-HCl buffer, pH 7.5 (Figure 2A). The small molar excess of Ni2+over UB- [His6]2, suggests tight binding of Ni2+to the Hise-tags which is consistent with the known binding affinity of recombinant Hise-tagged proteins to Ni-NTA resins. The measured width of a fiber is estimated to be ~ 4 ± 1 nm (Figure 2A). Since the effective diameter of a UB molecule is ~ 2.5 nm [Renatus, M., et al., Structure, 2006. 14(8): p. 1293-1302], this suggests that each fiber may be built from some form of UB dimers. Fiber lengths can exceed > 500 nm and, in some cases, 6- 8 fibers were observed to be aligned in parallel arrays (Figure 2A).

[0264] Polymer assembly was repeated with Zn2+under conditions similar to those used for Ni2+. Sheets, rather than fiber morphology, were observed. (Figure 2B; Figures 3A-C). With increased protein concentration, as well as with modified time and temperature of incubation, (Figure 2C), i.e., overnight incubation at 19 °C with Zn2+, imaging again showed sheet-like morphology. Measurement of the approximate width of the folded sheet tips gave 3-3.5 nm. The absolute requirement for divalent cations is demonstrated in Figure 2D, where ordered, one- or two- dimensional assembly is not observed. An additional control was performed: UB-[Hise]2 was replaced by UB-[His6]i, but with the same protocol followed as for Figure 2B. The absence of ordered, protein assemblies demonstrated the mandatory participation of both C- and N-terminal Hise-tags (Figures 4A-B).

[0265] Macromolecular Cas9-[Hise]2 polymerization promoted by ligand-divalent cation interaction: cryoTEM imaging

[0266] When UB-[Hise]2 was replaced by Cas9-[Hise]2, fibers > 500 nm long were observed in the presence of Ni2+(Figures 5A-B). Fiber width was estimated to be approximately 7 nm. Under similar conditions, but in the presence of Zn2+, approximately 500 nm x 500 nm two-dimensional sheets were generated (Figure 5C) with a folding pattern similar to that observed for UB-[His6]2. The approximate width of a folded tip was estimated to be 6.4 nm. In the absence of divalent cations, ordered assembly of Cas9-[Hise]2 into macromolecular polymers was not observed (Figure 5D).

[0267] Circular dichroism (CD) spectroscopy

[0268] Chiral peptide bonds of a protein give rise to characteristic CD spectral features at wavelengths < 240 nm that are sensitive to the secondary structure of the protein fold - alphahelix, beta-sheet, etc. The experimental CD spectrum of native Cas9 protein displays a negative ellipticity peak / positive ellipticity peak pattern, with two negative bands of nearly the same intensity, observed at 221 and 209 nm, accompanied by a positive peak at 196 nm [Halat et al., International Journal of Molecular Sciences, 2021. 22(6): p. 2937]. Recombinant Cas9-[His6]2, dissolved in DDW, with or without conjugation via Zn2+or Ni2+, show negative ellipticity peaks at - 224 nm and - 210 nm, and a positive ellipticity peak at - 194 nm (Figures 6A-D). Native mono-ubiquitin in PBS, pH 7, 37 °C displays three prominent ellipticity peaks: the first between 190 and 200 nm (positive), the second at - 205 nm (negative) along with a third , - 30% weaker, negative peak at -225 nm [Nguewa, P.A., et al., Chem Biodivers, 2005. 2(10): p. 1387-400]. Recombinant UB-[Hise]2, with or without conjugation via Ni2+or Zn2+, shows a positive ellipticity peak at 192 nm and negative peaks at 207 and 228 nm. Since the pKa of the imidazole side chain in histidine is - 6 while the pH of DDW varies between 5.5-6.9, it may be assumed that the pH during the CD measurements was sufficiently high to prevent protonation of the imidazole rings in the His-tags and consequently to promote chelation of the added metal, thereby leading to organized polymerization. Any changes that could be detected in the CD spectra of the recombinant [ HiseJ-taggcd proteins after 10 min incubation at 25 °C, when compared to the native spectra in the literature [see above] were considered to be insignificant (Figures 6A-D).

[0269] In summary, CD spectra indicated that, following incubation with Ni2+or Zn2+, both proteins remain folded and their secondary structure is preserved overall. The minor intensity changes in the spectra upon binding to the cations may indicate that the protein experiences small conformational changes following supramolecular polymerization.

[0270] Monitoring changes in electrophoretic mobility: SDS-PAGE and Native-PAGE

[0271] SDS-PAGE (Figure 7A) shows that, following SDS denaturation, both His-tagged proteins migrate on the gel under an electric field at or near the band positions expected for their molecular weights. However, evidence from non-denaturing Native PAGE electrophoresis (Figure 7B), where mobility is determined by conformation and charge, rather than molecular weight, shows that, even in the absence of metals, UB-[His6]2 apparently demonstrates a tendency to aggregate under the native gel conditions.

[0272] Rapid monitoring of solution changes by light microscopy

[0273] Light microscopy imaging, performed as described in the Methods section, demonstrates the following: (i) Ca2+or Mg2+cations, not known to bind to a Hise-tag, as do Zn2+and Ni2+, are not able to induce large scale precipitation or aggregation of UB-[His6]2 (Figure 8A); (ii) None of the four divalent cations, under identical working conditions, lead to protein precipitation when UB-[His6]2 is replaced by UB-[His6]i (Figure 8B). (iii) Low concentrations of EDTA, histidine or imidazole chelators, capable of competing with the Hise-tags of UB-[His6]2 for metal binding, can reverse the process and solubilize the preformed cation-bound UB-[His6]2 precipitates within 5 minutes (Figure 8C).

[0274] EXAMPLE 2

[0275] SUPRAMOLECULAR STRUCTURES OF HIS-TAGGED M-CHERRY

[0276] MATERIALS AND METHODS

[0277] SDS-PAGE (12%) and Native-PAGE (10%): Both mCherry derivative: (His)i-mCherry at 4.6 mg\mL and (His)2-mCherry at 14 mg / mL, were diluted with DDW to 0.15 mg / mL. While samples for SDS-PAGE analysis were reduced with beta-mercaptoethanol at 95°C for 10 minutes, samples for Native-PAGE were not reduced. Samples (18 pL) were loaded on both types of gels (with and without SDS) and separation as initiated.

[0278] Fluorescence spectrophotometer: Samples (500 pL) containing: 5 pM of the doubly Hise-tagged mCherry with or without 5 pM of ZnCh or NiCh in DDW were analyzed using the Agilent Cary Eclipse Fluorescence Spectrometer (Agilent, USA) equipped with a standard photomultiplier tube detector and FL Scan software. Measurements were conducted at a speed of 600 nm / min with both excitation and emission monochromator slits set to 5 nm. Each sample was scanned twice and all data was analyzed statistically using the FL scan software.

[0279] Circular dichroism (CD) analysis

[0280] Samples of 0.02 mg / mL (1 pM) of: (His) i -mCherry and 0.02 mg / mL (1 pM) (His)2- mCherry in DDW and the absence of Zn2+Ni2+( / '.<?., unconjugated state - control, [C]) or in their presence (at 1 pM) were dissolved to 0.05 mg / mL in double distilled water (DDW) were subjected to CD analysis using the Chirascan CD spectrometer (Applied Photophysics). CD spectra report ellipticity (9), proportional to the difference in absorbance of left and right circularly polarized light [9 = 3300° (AL~AR)] as a function of wavelength. A quartz cell of path length 0.1 cm was used for the measurements. The CD spectra were recorded with 2 nm bandwidth resolution in 1 nm steps at 25°C. CD spectra were corrected for baseline distortion by subtracting a reference spectrum of the corresponding solvent.

[0281] Scanning transmission electron microscopy (STEM)

[0282] Samples composed of 0.25 mg\mL (10 pM) (His)i-mCherry or (His)2-mCherry, in the absence of metals ( [C] ) or in the presence of 10 pM Zn2+or Ni2+were dissolved in double distilled water (DDW). These were loaded onto a carbon-coated copper grid Type-B (200 mesh) and dried overnight in a desiccator at 25 °C. Images were obtained using the UHR-MAIA3 TESCAN SEM with an In-Beam SE detector at HV 25 kV and magnification, 500 kX.

[0283] RESULTS

[0284] Analysis of the overexpressed (His)2-mCherry by SDS-PAGE led to the appearance of two bands at ~28 KDa and three bands at 16-18 KDa and <10 KDa under reducing conditions ( / '.<?., +beta-mercaptoethanol) (Figure 13A, lanes 2-3). A very similar pattern was observed with the mono Hisi-tagged mCherry ((His)i-mCherry) analog (Figure 13A, lanes 4-5). These findings suggested, that, the presence of two Hise-tags per mCherry monomer may affect its migration and split it into a doublet as this doublet was not observed with the (His)i-mCherry analog (Figure 13A, lanes 2-3 vs. 4-5). The presence of SDS (in SDS-PAGE) may be responsible for the lower Mw bands that appeared with both mCherry derivatives, as these bands were absent in Native- PAGE analysis where SDS is absent (Figure 13B, lanes 2-5). It was surprising to find that: (His)i- mCherry and (His)2-mCherry migrated on Native-gels to a distance representing 50-60 KDa implying their assembly into dimers (Figure 13B, lanes 2-5).

[0285] Fluorescent microscopy imaging showed, that, overnight incubation of 9 pM (His)i- mCherry at 8°C in the dark with equimolar concentration of Ni2+(in DDW), leads to spherical fluorescent particles (0.5-1 pm) (Figure 14B). The formation of these particles required the mandatory presence of Ni2+since in their absence, fluorescence was homogenously distributed (Figure 14B - inset). When Ni2+ions were replaced by Zn2+, elongated fluorescent structures appeared after 10 minutes (Figure 14C) and overnight incubation led to two distinct morphologies: spherical fluorescent particles (1-2 pm) and thicker and more elongated fluorescent assemblies (1- 2 pm x >10 pm) (Figure 14D). The greater particle diameter in the presence of Zn2+relative to that of Ni2+may derive from the ~20-fold greater binding affinity of Zn2+(Kd = 0.047 pM) towards the Hise-tag relative to that of Ni2+(Kd = 0.88 pM). Here again, a control experiment devoid of any added Zn2+resulted in even distribution of fluorescence (Figure 14D - inset).

[0286] These findings suggest that (a) added cations (Ni2+or Zn2+) participate in protein conjugation; (b) cation identity defines the size and morphology of the resulting architectures, (c) a [proteimcation] molar ratio of 1 : 1 is sufficient to trigger protein clustering, (d) these findings are consistent with the Native-PAGE analysis suggesting that (His)i-mCherry is dimeric (Figure 13B, lanes 4-5) and as such, can form macro-molecular assemblies upon metal addition.

[0287] Repetition of the protocol with (His)2-mCherry led to similar but not identical results. Fluorescent particles (0.5-1 pm) with Ni2+appeared after 10 minutes of incubation (Figure 15A) and not after overnight incubation as was observed with the (His)i-mCherry analog (Figure 14B). This finding may provide direct evidence for the contribution of a second Hise-tag per protein monomer that increases the rate of protein conjugation, the binding affinity or both. This argument is further supported by the observation of gigantic elongated structures >50 pm were generated after a longer incubation time (Figure 15B) that were not generated (His)i-mCherry under identical conditions (Figure 14B). The results with Zn2+differed from those with Ni2+. Fibers (~ 1 pm width x 5-10 pm length) were seen after 10 minutes (Figure 15C) with no significant change after overnight incubation (Figure 15D).

[0288] Since it seemed as if fluorescence is preserved in the presence of Ni2+and Zn2+, even after 14-96 hours of incubation at 8°C in the dark, it implied that mCherry's fluorophore site may had not been affected by the metals. However, to further support this hypothesis, the emission spectrum of (His)2-mCherry was measured in the absence or presence of cations used during the analysis with the fluorescence microscope (Figures 16A-B).

[0289] It was found that the emission spectrum of (His)2-mCherry devoid of any added cation was ~ 612 nm (Figure 16A-B) consistent with the previously reported one (610 nm) and that the presence of either: Ni2+or Zn2+at a concentration used for mCherry conjugation did not affect the emission spectrum even after 3 hours at 25 °C in the dark (Figure 16A-B). This provided quantitative direct evidence to the non-denaturing conditions in which (His)2-mCherry cluster.

[0290] The finding that the fluorophore site is not affected by the cations studied, does not exclude the possibility that the protein's secondary structure is not altered by Ni2+or Zn2+. It was therefore essential to perform circular dichroism (CD) analysis (Figure 17A-B). CD is a commonly employed, non-invasive analytical method used to assess changes in proteins' secondary structure. Comparison of the CD spectrum both mCherry analogs (z.e., (His) i -mCherry and (His)2-mCherry) without (control) or with Ni2+or Zn2+showed that their secondary structure is preserved in the presence of these metal ions even after 4 hours of incubation at 8 °C in the dark (Figure 17A-B).

[0291] Images of the resulting architectures at the nm scale were obtained with scanning transmission electron microscopy (STEM). Though this imaging technique is performed under dry conditions, it was unclear whether distorted protein-fibers and protein- sheets would be observed. The dry conditions in which this technique is performed, encouraged the use of DDW instead of buffers as the latter would precipitate and or crystallize during water evaporation and affect the resulting soft and fragile protein architectures generated in aqueous media.

[0292] Distorted-fibers and distorted- sheets were observed when 9 pM of (His)2-mCherry were incubated with 3 pM of Zn2+or Ni2+(Figures 18A-E). These were generated after overnight incubation at 8°C (in the dark) and at a [(His)2-mCherry:cation] molar ratio of 3:1 (Figure 18A- E). Very similar protein-fibers and protein- sheets were obtained at a 1:1 molar ratio (not shown). When no metal was added, fibers and sheets were not observed (Figure 18F).

[0293] The observed 3:1 molar ratio suggests, that, several Hise-tags may be bound to a central, single Zn2+or Ni2+cation. This speculation might be consistent with the observation that (a) Zn2+can from tetrahedral or octahedral coordination with 4 or 6 imidazoles (b) two histidine or imidazole end groups may be complexes by a single Zn2+, Ni2+, Cu2+or Co2+, but clearly cannot allow determination of the ratio in the observed mCherry fibers or sheets.

[0294] Since longer incubation times (e.g., 4 days at 8°C in the dark) at the same 3: 1 ratio, did not generate different structural morphologies, it was concluded that macromolecular assembly requires hours (z.e., overnight) of incubation rather than days.

[0295] Fiber's length with both metals was at the micron size, exceeding 20 pm (Figure 18A-D). Protein- sheets had similar dimensions (Figure 18E). As the hydrodynamic radius of mCherry ranges between 2.33-2.5 nm it follows, that, the number of (His)2-mCherry present within a 10 pm fiber is estimated to be a few thousands and several millions in 10 pm x 10 pm sheets.

[0296] Very similar findings were observed when the same protocol was applied on the mono Hise-tagged mCherry analog (His)i-mCherry (Figure 19A-D). Fibers and sheets of (His)i- mCherry) were found with both cations (Figure 19A-C) and none, in the absence of zinc or nickel (Figure 19D). These finding derive from the dimerization state in which (His) i -mCherry exists Figure 13B).

[0297] Process reversibility was studied by the addition of chelators (e.g., EDTA, histidine, imidazole), after protein-assembly (Figures 20A-B). These, were expected to compete on the complexed cation, dissociate protein-assemblies and generate a homogenous distribution of fluorescence. Indeed, 5 minutes after the addition of 5 mM of either: EDTA, histidine or imidazole (all at pH 7) the globular or linear-fluorescent assemblies formed after overnight incubation of 10 pM of (His)2-mCherry and equimolar concentration of either Zn2+or Ni2+led to an even distribution of red fluorescence (Figures 20A-B).

[0298] Complete dissolution was achieved with EDTA and histidine while imidazole led to partial disintegration (Figures 20A-B). These findings may derive from differences in the binding affinity of the studied chelators to Zn2+or Ni2+. While EDTA and histidine function as a hexadentate- ligand and a tridentate-ligand respectively towards Zn2+or Ni2+(EDTA: Ni =18.6, Zn = 16.5), imidazole represents a monodentate-ligand and hence, binds such cations with a lower affinity.

[0299] Since it is likely, that, complexes between mCherry's Hise-tags and Zn2+or Ni2+are responsible for protein conjugation, the present inventors wondered what would be the pH range that would still support protein assembly. Therefore, protein conjugation was repeated in the presence of 50 mM Na citrate at a wide range of pH values (z.e., 7-3). It was also assumed, that, the histidine side chain (z.e., the imidazole ring) would participate in metal chelation, only if the imidazole ring is not protonated and this is determined by the system pH. Since the pKa of the imidazole ring in histidine's is ~ 6, then only 50% of His side chains are free to chelate Zn2+or Ni2+at pH 6 for example.

[0300] It was found, that, conjugation of (His)2-mCherry with Zn2+was successful at pH 7 and 6, but became less effective at pH 5 and totally ineffective at pH 4 as homogenous distribution of fluorescence was clearly observed (Figure 21). At pH 3, no fluorescence was identified, rather darkness that likely derives from complete destruction of the protein's fluorophore site knows to be significantly affected already at pH 4.5. Thus, the presented strategy seems to be limited to acidic conditions, not lower than pH 5.

[0301] 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.

[0302] It is the intent of the applicant(s) that all publications, patents and patent applications referred to in this specification are to be incorporated in their entirety by reference into the specification, as if each individual publication, patent or patent application was specifically and individually noted when referenced that it is 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 generating a supramolecular structure comprising:(a) contacting a divalent or trivalent metal cation with at least one protein-based molecule comprising at least two histidine tags under conditions that generate the supramolecular structure; and(b) isolating the supramolecular structure.

2. The method of claim 1, wherein the supramolecular structure is a nanofiber or microfiber.

3. The method of claim 1, wherein the supramolecular structure is a nanosheet or microsheet.

4. The method of claim 1, wherein the supramolecular structure is not a cyclic structure.

5. The method of any one of claims 1-3, wherein said metal cation is selected from the group consisting of Zinc (Zn2+), Nickel (Ni2+), iron (Fe2+or Fe3+), or cobalt (Co2+).

6. The method of any one of claim 1-3, wherein said divalent metal cation are Zinc (Zn2+) or Nickel (Ni2+).

7. The method of any one of claims 1-6, wherein said protein-based molecule is a monomeric polypeptide.

8. The method of any one of claims 1-6, wherein said protein-based molecule is a dimer or trimer.

9. The method of claim 7, wherein a first histidine tag of said at least two histidine tags is positioned at the N-terminus of said polypeptide and a second histidine tag of said at least two histidine tags is positioned at the C-terminus of said polypeptide.

10. The method of any one of claims 1-9, wherein each of said at least two histidine tags comprise at least 4 consecutive histidines.

11. The method of any one of claims 1-6, wherein said at least one protein-based molecule is water soluble.

12. The method of any one of claims 1-6, wherein said at least one protein-based molecule is a membrane protein.

13. The method of any one of claims 1-11, wherein said at least one protein-based molecule is a fluorescent polypeptide.

14. The method of any one of claims 1-12, wherein said at least one protein-based molecule is an enzyme or receptor.

15. The method of any one of claims 1-14, wherein said at least one protein-based molecule is devoid of an amino acid sequence as set forth in SEQ ID NO: 8.

16. The method of any one of claims 1-15, wherein said at least one protein-based molecule comprises at least two protein-based molecules, each comprising said at least two histidine tags.

17. The method of claim 16, wherein one of said at least two non-identical proteinbased molecule is an enzyme label and a second of said at least two non-identical protein-based molecule binds specifically to an Fc domain of an antibody.

18. The method of claim 17, wherein said second protein-based molecule is Protein G or Protein A.

19. A supramolecular structure generated according to the method of any one of claims 1-18.

20. A composition comprising a purified supramolecular structure which comprises at least two repeating units, each repeating unit being represented by the formula P-D-P-D, whereinP is a protein-based molecule comprising at least two histidine tags; andD is a divalent or trivalent ion, wherein each protein-based molecule in the structure is non-covalently bound to at least one other protein-based molecule via said divalent or trivalent ion.

21. The composition of claim 20, wherein at least one said divalent or trivalent ion of the structure coordinates non-covalent binding of 2-6 said protein-based molecules.

22. The composition of claim 20, wherein at least one said divalent or trivalent ion of the structure coordinates non-covalent binding of four said protein-based molecules.

23. The composition of claim 20, wherein said supramolecular structure is a nanofiber, a microfiber, a nanosheet or a microsheet.

24. The composition of claims 20 or 23, being essentially devoid of structures composed of P-D-P.

25. The composition of any one of claims 20-24, wherein said metal cation is selected from the group consisting of Zinc (Zn2+), Nickel (Ni2+), iron (Fe2+or Fe3+), or cobalt (Co2+).

26. The composition of any one of claim 20-24, wherein said metal cation are Zinc (Zn2+) or Nickel (Ni2+).

27. The composition of any one of claims 20-26, wherein said at least one protein based molecule is a polypeptide monomer.

28. The composition of any one of claims 20-26, wherein said at least one proteinbased molecule is a dimer or trimer.

29. The composition of any one of claims 27, wherein a first histidine tag of said at least two histidine tags is positioned at the N-terminus of said polypeptide monomer and a second histidine tag of said at least two histidine tags is positioned at the C-terminus of said polypeptide monomer.

30. The composition of any one of claims 20-29, wherein each of said at least two histidine tags comprise at least 4 consecutive histidines.

31. The composition of any one of claims 20-30, wherein said protein-based molecule is water soluble.

32. The composition of any one of claims 20-31, wherein said protein-based molecule is a fluorescent polypeptide.

33. The composition of any one of claims 20-31, wherein said protein-based molecule is a biomolecule binding protein.

34. The composition of any one of claims 20-31, wherein said protein-based molecule is not an amyloid protein.

35. The composition of any one of claims 20-31, wherein said protein-based molecule is not an amphiphilic protein.

36. The composition of any one of claims 20-31, wherein said protein-based molecules of said supramolecular structure are not connected via amphiphilic peptides.

37. The composition of any one of claims 20-31, wherein said at least one proteinbased molecule is an enzyme.

38. The composition of any one of claims 20-31, wherein said at least one proteinbased molecule is a receptor.

39. The composition of any one of claims 20-38, composed of at least two non-identical protein-based molecules, each comprising said at least two histidine tags.

40. The composition of claim 39, wherein one of said at least two non-identical proteinbased molecules is an enzyme label and a second of said at least two non-identical protein-based molecules binds specifically to an FC domain of an antibody.

41. The composition of claim 40, wherein said second protein-based molecule is Protein G or Protein A or a fragment thereof.

42. A method of isolating a biomolecule from a solution comprising:(a) contacting the biomolecule with the composition of claim 33 under conditions that allows binding of said biomolecule to said biomolecule binding protein; and(b) removing said supramolecular structure from said solution.

43. A biomolecule affinity agent comprising a supramolecular structure which comprises at least two repeating units, each repeating unit being represented by the formula P-D- P-D, whereinP is a detectable protein-based molecule comprising at least two histidine tags; andD is a divalent or trivalent ion, wherein at least one detectable protein-based molecule in the structure is non-covalently bound to a second detectable protein-based molecule via said divalent or trivalent ion, wherein the supramolecular structure further comprises a protein that binds directly or indirectly to the biomolecule, said protein being non-covalently bound to said detectable proteinbased molecule via said divalent or trivalent ion.

44. The biomolecule affinity agent of claim 43, wherein said protein binds to an FC region of an antibody.

45. The biomolecule affinity agent of claims 43 or 44 wherein a ratio of said detectable protein-based molecule: said protein is at least 3:1.

46. A method of detecting a biomolecule comprising:(a) contacting said biomolecule with said biomolecule affinity agent of any one of claims 43-45, under conditions which allow binding of said biomolecule to said biomolecule affinity agent; and(b) detecting said detectable protein-based molecule.

47. The method of claim 46, further comprising contacting said biomolecule with an antibody prior to step (a).

48. An article of manufacture comprising a solid surface attached to the composition of any one of claims 20-41.

49. The article of manufacture of claim 48, being a medical device.

50. The article of manufacture of claim 48, being a detection device.

51. A method of performing an enzymatic reaction comprising contacting the composition of claim 37 with starting components of an enzymatic reaction under conditions that allow the enzyme to catalyze the enzymatic reaction, thereby performing the enzymatic reaction.

52. A method of generating an antibody in a subject comprising administering to the subject the composition of any one of claims 20-38, thereby generating the antibody.

53. The method of claim 52, wherein the composition is devoid of an additional adjuvant.

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