WO2013172701A1

WO2013172701A1 - Fusion proteins capable of both ph and temperature induced self-assembly into nanocapsules

Info

Publication number: WO2013172701A1
Application number: PCT/NL2013/000028
Authority: WO
Inventors: Mark VAN ELDIJK; Jan Van Hest
Original assignee: Stichting Dutch Polymer Institute
Priority date: 2012-05-14
Filing date: 2013-05-14
Publication date: 2013-11-21

Abstract

The invention relates to a protein comprising a first polypeptide segment capable of forming a nanocapsule by assembling with a plurality of other polypeptide segments that are also capable of forming the nanocapsule and a second polypeptide segment capable of undergoing an inverse temperature transition, the segment comprising two or more monomeric units selected from the group of tetrapeptides, pentapeptides and hexapeptides. In the exemplified chimeric protein, the first polypepide fragment is the cowpea chlorotic mottle virus (CCMV) capsid protein, and the second polypeptide comprises monomeric units derived from elastin that are known as elastin-like polypeptides.

Description

FUSION PROTEINS CAPABLE OF BOTH PH AND TEMPERATURE INDUCED SELF-ASSEMBLY INTO NANOCAPSULES

The invention relates to a protein capable of forming a nanocapsule, to a method of preparing such proteins, to a composition comprising nanocapsules that comprise such proteins, to a method for preparing such composition and to the use of such nanocapsules.

Self-assembled nanocapsules have been studied intensively over the past years because of their release and storage capacity, which can be of use in a range of applications, varying from drug delivery vehicles to

nanoreactors. Different types of materials have been used to generate nanocapsules, such as lipids, polymers, proteins, or combinations thereof. Proteins are particularly useful building blocks since proteins are capable of forming well-defined structures by their intricate three-dimensional folding. It has been demonstrated that well-defined nanocapsules can be prepared from protein constituents in a self-assembling manner.

Nature is known to make use of a variety of protein-based

nanocapsules, of which viruses constitute a prominent class. A virus is a naturally occurring protein cage (a nanocapsule) that is assembled from a plurality of protein-subunits so as to encapsulate the virus's genetic material for storage and transport. The number of protein-subunits in one virus particle may be from several tens to several hundreds, depending on the type of virus. In the art, the protein cage of a virus is named a "capsid". Analogously, a viral protein-subunit capable of forming a capsid is named "capsid protein" (CP).

It has been found that many virus proteins are also capable of assembling a nanocapsule without encapsulating their natural contents. For example, Cowpea Chlorotic Mottle Virus (CCMV) can be disassembled and reassembled by adjusting the pH, even after removal of its viral RNA. At higher pH (7.5) empty CCMV nanocapsules dissociate into dimers of protein protein-subunits and at lower pH (5.0) these dimers associate to form the cage via self-assembly. Nanocapsules assembled from proteins from CCMV have been shown to be useful as nanoreactors or as template for constrained synthesis of nanomaterials. However, control over the formation of nanocapsules from protein sub-units can usually only be achieved by changing the pH in a certain range (see e.g. J. A. Speir, et al in Structure, 1995, 3, 63-78 for CCMV and M. Cuillel et al in, J. Mol. Biol., 1983, 164, 589- 603 for Brome Mosaic Virus (BMV)). This limits the application of the

nanocapsules that are assembled from such proteins. Moreover, it is

undesirable that substances need to be added to a composition comprising the nanocapsules in order to have control over the formation of

nanocapsules, as is usually the case when inducing a change in the pH. The addition of a substance is invasive to the composition comprising the

nanocapsules, since the opposite action (i.e. instant withdrawal of only the specific substance) is usually too complicated to perform or it is incompatible with the desired application of the nanocapsules.

It is therefore an object of the present invention to provide a protein capable of forming a nanocapsule, wherein the formation of the nanocapsules can be controlled in a manner that is reversible. It is in particular an object of the invention to exert the control in a thermally-responsive manner.

It is a further object of the invention to provide a protein capable of forming a nanocapsule, wherein it is possible to tune the temperature range in which the disassembly-reassembly process of the nanocapsule occurs. This means that by proper design of the protein or by choosing the proper

conditions for the composition in which the disassembly-reassembly process occurs, a nanocapsule is prepared that has the desired temperature range in which it reversibly disassembles and reassembles.

It has now been found that this objective can, at least in part, be reached by functionalizing a polypeptide that is capable of forming a

nanocapsule with a polypeptide that has other specific properties.

Accordingly, the present invention relates to a protein comprising - a first polypeptide segment capable of forming a nanocapsule by

assembling with a plurality of other polypeptide segments that are also capable of forming the nanocapsule;

- a second polypeptide segment comprising two or more monomeric units selected from the group of tetrapeptides, pentapeptides and hexapeptides.

In particular, the second polypeptide segment is capable of For the purpose of the invention, with a polypeptide is meant a linear chain of two or more subsequent amino acids. The amino acids in a polypeptide are connected to each other through peptide bonds. An oligopeptide is meant to indicate a polypeptide wherein the number of amino acids is relatively small, in particular from 2 to 10. In the context of the invention, a polypeptide segment is a sequence of amino acids that forms a particular part of the chain of a polypeptide. A protein comprises one or more polypeptides arranged to facilitate a certain (biological) function.

For the purpose of the invention, with a nanocapsule is meant a container of nanometer dimensions. Each dimension of a nanocapsule is usually in the range of 5 to 500 nm. The shape of a nanocapsule is usually essentially spherical, or cylindrical (rod-like).

The first polypeptide segment in a protein of the invention is as such capable of forming a nanocapsule by assembling with a plurality of other polypeptide segments that are also capable of forming the nanocapsule. Such a nanocapsule is thus an assembly of a plurality of these polypeptide segments. In particular, the first polypeptide segment in a particular protein of the invention is as such capable of forming a nanocapsule by assembling with a plurality of first polypeptide segments of other proteins of the invention. Such a nanocapsule is thus an assembly of a plurality of first polypeptide segments. In case all first polypeptide segments are the same, the

nanocapsule that is assembled from them essentially consists of a plurality of first polypeptide segments.

The first polypeptide segment in a polypeptide of the invention usually has a mass in the range of 5-200 kD.

The first polypeptide segment in a protein of the invention may be of viral origin. This means that the first polypeptide segment is derived from the capsid protein of a virus and bears great similarity with the capsid protein of that virus, or at least with a part of the capsid protein of that virus. The first polypeptide segment may for example have at least 50%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the capsid protein of that virus or to a part of the capsid protein of publicly available sequence comparison algorithms, known to the skilled person.

A polypeptide segment of viral origin is not necessarily obtained directly from a virus, but may also be obtained by, for example, bacterial expression. The first polypeptide segment may for example be derived from CCMV. In the event that the first polypeptide segment is derived from the capsid protein of a virus, that virus preferably has the N- and/or the C- terminus at the interior of its capsid.

The second polypeptide segment in a protein of the invention is capable of undergoing an inverse temperature transition, and comprises two or more monomeric units selected from the group of tetrapeptides, from the goup of pentapeptides, or from the group of hexapeptides. The number of monomeric units is usually in the range of 2-160. Preferably it is in the range of 5-50, more preferably it is in the range of 5-25, in the range of 5- 5 or in the range of 6-12. In particular, the number of monomeric units is 9.

The monomeric units in a certain polypeptide segment are usually identified by the characteristic that at least two amino acid residue positions in a monomeric unit are occupied by the same amino acid residue in all monomeric units in that segment. For example, when tetrapeptides of the formula Kaa-Laa-Maa-Naa are present in a certain segment (wherein Kaa is the amino acid at the first position, Laa the amino acid at the second position, Maa the amino acid at the third position, and Naa the amino acid at the fourth position), then two of these amino acids (including their position) remain the same throughout all tetrapeptide monomeric units present in that polypeptide segment, while the other two may vary (e.g. when Kaa and Maa remain unchanged, Laa and Naa may vary).

The oligopeptide monomer units in the second polypeptide segment are in particular derived from elastin. Elastin is a protein in connective tissue that is elastic and allows many tissues (e.g. lungs, intestines and skin) in the human or an animal body to resume their shape after stretching or

contracting. Elastin contains pentapeptide monomer units of the formula Val- Pro-Gly-Val-Gly. In the art, when a polypeptide is said to be derived from containing oligopeptide monomeric units derived from Val-Pro-Gly-Val-Gly are also named "elastin-like polypeptides" (ELP's).

The second polypeptide segment in a protein of the invention may in particular comprise two or more pentapeptide monomeric units of Xaa-Pro- Yaa-Zaa-Gly, wherein, for each pentapeptide monomeric unit, Xaa is independently chosen from the amino acids isoleucine and valine, Yaa is independently chosen from the amino acids glycine and alanine and Zaa is independently chosen from the group of alpha-alkylated alpha amino acids and glycine. More in particular, in pentapeptide monomeric units of Xaa-Pro- Yaa-Zaa-Gly, the amino acid Xaa is valine and the amino acid Yaa is glycine. The monomeric unit in this case is thus Val-Pro-Gly-Zaa-Gly.

In the art, pentapeptide monomeric units of Val-Pro-Gly-Zaa-Gly are usually described using the notation ELP[XjY_jZ_k-n]. The capital letters between the brackets indicate the single letter amino acid code for the Zaa replacing residue in the pentapeptide monomeric units Val-Pro-Gly-Zaa-Gly. The subscript represents the overall-ratio of the different Zaa residues present in all Val-Pro-Gly-Zaa-Gly pentapeptide monomeric units of the second polypeptide segment. The letter n represents the total number of Val- Pro-Gly-Zaa-Gly pentapeptide monomeric units in the second polypeptide segment.

The second polypeptide segment in a protein of the invention is capable of undergoing an inverse temperature transition. With this

characteristic of a polypeptide (or a segment thereof) is meant the following. Below its transition temperature (T_t), the polypeptide is highly soluble in aqueous solutions. However, when the temperature is raised above T_t, the hydrated polypeptide chains collapse and aggregate under the influence of hydrophobic groups on the polypeptide chain. A non-soluble ELP-rich phase is formed, which separates from the aqueous solution. When the temperature is lowered to below the T_t, the polypeptide in the separated ELP-rich phase redissolves into the aqueous solution. Accordingly, the switch between the extended water-soluble and the collapsed hydrophobic state is reversible.

The transition can in principle also be triggered by other stimuli than said to possess stimulus-responsive properties. Such stimuli are usually also considered as a means to change the T_t of the respective polypeptide; the main characteristic property of the transition is in all cases that it is an inverse temperature transition.

In particular, for polypeptides derived from elastin, the transition temperature T_t between both states can be influenced by the following stimuli; (1) by changing the Zaa residue(s) in one or more pentapeptide monomeric unit(s); (2) by changing the number of pentapeptide monomeric units in the polypeptide; (3) by changing the protein concentration ;(4) by changing the concentration of solutes such as salt in the composition wherein the

polypeptide that comprises the pentapeptide monomeric units is present.

A protein of the invention comprises both types of polypeptide segments. Both segments are usually connected to each other, preferably via covalent bonding. They can be connected directly to each other by one covalent bond, in particular a peptidic bond. Both segments can also be connected to each other by a linker, in particular a third polypeptide segment, wherein each end of the linker is connected to one of their ends. When both segments are connected to each other, they are usually part of one and the same chain.

A protein of the invention appears to possess a beneficial

combination of the functions and properties of the two polypeptide segments. On the one hand, the first polypeptide segment provides the protein-subunits for building a well-defined structure and also still possesses the property to actually create such well-defined structures from these protein-subunits via self-assembly. On the other hand, the second polypeptide segment has retained its capability to undergo an inverse temperature transition (its stimulus-responsive character) when it is in a combination with the first segment. This combination of functions and properties of both polypeptide segments gives a protein of the invention its unique properties, in particular the property that nanocapsules may be formed from a protein of the invention upon applying a stimulus for ELP.

It has been found that with a protein of the invention, two kinds of of a first kind, generated via pH-induced self-assembly and (2) nanocapsules of a second kind, generated via temperature-induced self-assembly.

The first kind of nanocapsules is obtained via "conventional" self- assembly, i.e. self-assembly that is analogous to the self-assembly that would occur if the first polypeptide segment was not functionalized with the polypeptide capable of undergoing an inverse temperature transition (a stimulus-responsive polypeptide).

In the generation of nanocapsules of the second kind, the capability of polypeptides to undergo an inverse temperature transition plays an important role in the architecture that is formed. The process of self-assembly in the second type is therefore different from that of the first type, which results in nanocapsules of a different kind.

The conditions during the assembly process determine which of the two kinds of nanocapsules is formed. The formation of the first kind of nanocapsules is governed by the pH of the composition comprising a protein of the invention: at higher pH the nanocapsules dissociate into protein building blocks and at lower pH these building blocks associate to form the nanocapsule via self-assembly. When, however, at a higher pH (i.e. after dissociation of the nanocapsules into smaller fragmets) the temperature of the composition is raised to above the transition temperature T_t, nanocapsules of the second kind are formed.

An advantage of a protein of the invention is thus that it allows the temperature-induced self-assembly and disassembly of nanocapsules.

A further advantage is that the temperature of the induced self- assembly and disassembly of nanocapsules can be tuned by changing the concentration of the protein and/or the solute, and/or by changing the polypeptide segments by choosing specific oligopeptide monomeric units.

The finding that the functionalization of an architecture-inducing polypeptide with a polypeptide capable of undergoing an inverse temperature transition results in the conservation of the functions and properties of both polypeptides is surprising, since the functionalization of a polypeptide with a polypeptide capable of undergoing an inverse temperature transition often structure. Or, in the case that dissolved particles do form, they appear to be fragments that have a high diversity in structure.

In an embodiment, a protein of the invention also comprises an histidine tag, which is either an N-terminal histidine tag or a C-terminal histidine tag. Such a tag usually comprises 3 to 9 histidine residues, preferably it comprises 6 histidine residues. Such a histidine tag is denoted as Ηχ, wherein x is the number of histidine residues.

A histidine tag is usually connected to the second polypeptide segment ELP[XjYjZk-n]. For example, a polypeptide segment resulting from the combination of (1 ) a polypeptide segment ELP[XiYjZ_k-n] and (2) an N- terminal histidine tag comprising 6 histidine residues is denoted as

H₆-ELP[XiY_jZ_k-n].

A histidine tag in a protein of the invention usually serves to simplify the purification process of the protein after its synthesis. It has been shown that the unique properties of a protein of the invention do not rely on the presence of the histidine tag. However, a possible influence of the tag on the assembly properties of the protein cannot be excluded.

In a specific embodiment of the invention, the first polypeptide segment is derived from CCMV and the second polypeptide segment is H₆-ELP[V₄L₄G 9]. The encoded amino acid sequence of the protein comprising both segments is provided below as SEQ ID NO.1.

SEQ ID NO.1 :

G H H H H HHVPGVGVPGLGVPGVGVPG LGVPGVGVPG LG VPGGG VPG VGV PGLGLEWQPVIVEPIASGQGKAIKAWTGYSVSKWTASCAAAEAKVTSAITIS LPNELSSERNKQLKVGRVLLWLGLLPSVSGTVKSCVTETQTTAAASFQVAL AVADNSKDWAAMYPEAFKGITLEQLTADLTIYLYSSAALTEGDVIVHLEVEH VRPTFDDSFTPVY. An aqueous composition comprising the polypeptide of SEQ ID NO.1 exhibits the association-dissociation behavior under the different pH and temperature as described above. Nanocapsules assembled from the Klug triangulation number (T) = 3 (D.L.D. Caspar, A. Klug, Cold Spring Harb Sym 1962, 27, 1-24)). This means that the protein-subunits have largely the same position and orientation in both assemblies. A different kind of nanocapsules is assembled when the composition is brought to a pH of 7.5 and is then exposed to a temperature above the transition temperature T_t. The diameter of these nanocapsules is 18 nm (T=1).

Both kinds of nanocapsules were shown to be monodisperse, since essentially no other structures comprising a plurality of proteins of the invention could be observed. It was also demonstrated that both kinds of nanoparticles in principle do not exist simultaneously in one composition.

The invention further relates to a composition comprising

nanocapsules and an aqueous solvent, each nanocapsule comprising a plurality of proteins according to the invention. It is herewith understood that the plurality of proteins indicates the presence of a plurality of the same protein particles. In particular, the nanocapsules comprise proteins having a polypeptide segment derived from the Cowpea Chlorotic Mottle Virus.

In an embodiment, the nanocapsules comprise a physical entity, for example a pharmaceutically active compound and/or an enzyme.

In a nanocapsule that is formed from a protein of the invention, the first polypeptide segments generally have their N- and/or C-termini at the interior of the nanocapsule. In particular, in a nanocapsule that is formed from proteins wherein the N-terminus of the first polypeptide segment is connected to the C-terminus of the second polypeptide segment, the N-terminus of the first polypeptide segment is at the interior of the nanocapsule. This ensures that the second polypeptide segment is also at the interior of the

nanocapsule, i.e. it is inside the nanocapsule and not outside the

nanocapsule. It is contemplated that a self-assembled shell is formed within the nanocapsule from a plurality of second polypeptide segments that are at the interior of the nanocapsule, and that this assembly is responsible for (1 ) the different size from the pH-induced nanocapsules and (2) the unique temperature responsive properties of the temperature-induced nanocapsules. Likewise, in a nanocapsule that is formed from polypeptides wherein the C- the second polypeptide segment, the C-terminus of the first polypeptide segment is in particular at the interior of the nanocapsule.

The invention further relates to a method for preparing a protein, comprising

- constructing a vector coding for bacterial expression of a polypeptide

capable of forming a nanocapsule by assembling with a plurality of other polypeptides that are also capable of forming the nanocapsule, then; inserting DNA into the vector encoding for bacterial expression of a polypeptide capable of undergoing an inverse temperature transition and comprising two or more monomeric units selected from the group of tetrapeptides, pentapeptides and hexapeptides, then;

Transforming the resulting plasmid into a bacterium, then;

Expressing the protein, and then;

Purifying the expressed protein.

In a method of the invention, the plasmid is usually transformed into

E. coli cells, for example E. coli BLR(DE3)pLysS cells.

In an embodiment, the DNA encodes for bacterial expression of a polypeptide comprising two or more monomeric units derived from elastin.

In a further embodiment, the DNA encodes for bacterial expression of a polypeptide comprising two or more pentapeptide monomeric units of Xaa- Pro-Yaa-Zaa-Gly, wherein, for each pentapeptide monomeric unit, Xaa is independently chosen from the amino acids isoleucine and valine, Yaa is independently chosen from the amino acids glycine and alanine and Zaa is independently chosen from the group of alpha-alkylated alpha amino acids and glycine. In particular, in the two or more pentapeptide monomeric units of Xaa-Pro-Yaa-Zaa-Gly, the amino acid Xaa is valine and Yaa is glycine.

In a further embodiment, the first polypeptide segment is of viral origin, in particular it is derived from the Cowpea Chlorotic Mottle Virus.

The invention further relates to a method for preparing a composition comprising nanocapsules. The method comprises mixing the protein of the invention with an aqueous solvent, followed by incubating the resulting composition. Optionally, additional matter that is present in the solution is this way, nanoparticles can be obtained that contain a specific compound, for example a pharmaceutically active compound.

As described above, two kinds of nanocapsules can be formed in a self-assembling manner: (1) nanocapsules of a first kind, generated via pH- induced self-assembly and (2) nanocapsules of a second kind, generated via temperature-induced self-assembly.

To obtain a composition with nanoparticles of the first kind, the protein of the invention is mixed with an aqueous solution of a pH at which pH-induced nanoparticles are formed in a self-assembling manner. The temperature of the composition during the preparation of the composition does not need to be changed.

To obtain a composition with nanoparticles of the second kind, the protein of the invention is mixed with an aqueous solution of a pH at which pH-induced nanoparticles disassemble. In particular, the pH is within the range wherein the temperature-induced self-assembly can occur. In addition, the temperature of the composition during incubation is equal to or higher than the transition temperature for switching between the hydrophilic state and the hydrophobic state of the second polypeptide segment in the protein.

In a particular embodiment, the nanocapsules comprise a protein having a polypeptide segment derived from the Cowpea Chlorotic Mottle Virus. A composition comprising nanoparticles of the first kind is then obtained when the pH of the aqueous solution is 5.0 or lower. All steps of the preparation are performed at room temperature. A composition comprising nanoparticles of the second kind is obtained when the pH of the aqueous solution is 7.5 or higher, when NaCI is present in a concentration of at least 1.8 M and when the temperature of the composition during incubation is 35 °C.

The formation of nanoparticles of the second kind from a protein of the invention is schematically shown in Figure 1. The protein unit is shown in the left-hand side of this figure. It comprises the first polypeptide segment in its upper part (as a folded chain) and the second polypeptide segment in its lower part (as a chain that is less folded). A cryo-EM reconstruction of the interior of the nanocapsule. It is in particular shown that the second polypeptide segment is at the interior of the nanocapsule.

When a protein of the invention is exposed to aqueous conditions that do not allow the formation of any of the two kinds of nanoparticles, the protein is usually present as a dimer in those aqueous conditions (for the sake of clarity, Figure 1 displays the protein monomer).

Any of the two kinds of nanoparticles may form from the dimer of the protein of the invention. It is not necessary for the formation of any of the two kinds of nanoparticles that the other kind has previously existed in the aqueous solution.

The invention further relates to the use of a composition according to the invention for the controlled release of encapsulated molecules or as template for the synthesis of nanoporous materials.

The controlled release of encapsulated molecules is made possible by the property that nanocapsules of the invention can be disassembled in a controlled manner. The controlled release of encapsulated molecules can for example be used for the purpose of drug delivery in living organisms. It can also be used to initiate diagnostic tests wherein it is required that enzymes are released uniformly and in a controlled manner through the medium (e.g. in ELISA).

Nanocapsules that are assembled from a polypeptide with the amino acid sequence of SEQ ID NO. 1. have a diameter of about 18 nm or about 28 nm, depending on the assembly conditions. These sizes makes these nanocapsules particularly useful as a template for the synthesis nanoporous materials.

EXAMPLES Materials and methods

Protein concentrations were determined using a Cary 50 Cone theoretical extinction coefficients. [S. C. Gill, P. H. von Hippel, Anal Biochem 1989, 182, 319-326]

TEM grids (FCF200-Cu, EMS) were glow-discharged using a

Cressington Carbon coater and power unit. 5 μΙ_ sample (0.2 mg/mL) was applied to the glow-discharged grid and was incubated for 1 minute. Then the sample was removed carefully using a filter paper and the grid was allowed to dry for at least 15 min. Subsequently, the grid was negatively stained by applying 5 μΙ_ of 2% uranyl acetate in water. The staining solution was removed after 15 seconds and the grid was again allowed to dry for at least 15 min. The samples were analyzed on a JEOL JEM-1010 TEM (Jeol, Japan).

DLS experiments were performed on a Zetasizer Nano S (Malvern Instruments Ltd, England). All samples were first purified by size exclusion chromatography (SEC).

Restriction enzymes, ligase and kinase were obtained from New

England Biolabs. The DNA oligos were synthesized by Biolegio (Nijmegen, The Netherlands).

The wildtype capsid protein (WT-CP) was provided by J.J.L.M.

Cornelissen from University of Twente, The Netherlands.

Two types of buffers were used, a pH 5.0 buffer (50 mM NaOAc, 500 mM NaCI, 10 mM MgCI₂, 1 mM EDTA, pH 5.0 set with HCI) and a pH 7.5 buffer (50 mM TrisHCI, 500-2500 mM NaCI, 10 mM MgCI₂, 1 mM EDTA, pH 7.5 set with HCI). The NaCI concentration in the last buffer was varied between 500 and 2500 mM. Prior to use, all buffers were filtered over a Millipore 0.2 μηι filter and for the SEC-MALLS analysis, the buffers were also degassed.

Cloning of ELP-functionalized Capsid Protein The pET-15b-H₆-CP vector coding for bacterial expression of the histidine-tagged CCMV capsid protein was previously constructed as described by Minten et al.[ I. J. Minten, L. J. A. Hendriks, R. J. M. Nolte, J. J. DNA oligos with a complementary overhang were designed. These two sets of oligos were annealed and the resulting insert encoded for a histidine-tag, the ELP[N ₄UGi-9] and a short fragment of the CP with a 5' Ncol and a 3' Agel restriction site. First, the pET-15b-H₆-CP vector was digested with Ncol- HF™ and Agel-HF™, and then the product was purified by agarose gel electrophoresis. Subsequently, the annealed inserts were ligated into the digested vector. The linear product was again purified by agarose gel electrophoresis and then it was phosphorylated and ligated to yield the pET- 15b-H₆-ELP[V₄l-4Gi-9]-CP(AN26). This plasmid was transformed into E. coli XL1-Blue cells and then the DNA was extracted and the sequence was confirmed by DNA sequencing. The plasmid was transformed into E. coli BLR(DE3)pLysS cells (Novagen, MERCK), which were used for the

expression of H6-ELP[V₄L₄G₁-9]-CP(AN26). Expression and purification of capsid proteins

For a typical expression, 100 ml_ 2xYT medium, supplemented with ampicillin (100 mg/L) and chloroamphenicol (50 mg/L), was inoculated with a single colony of E. coli BLR(DE3)pLysS containing pET-15b-H6-CP or pET- 15b-H6-ELPr ₄L₄G₁-9]-CP(AN26) and was incubated at 30 °C overnight. This overnight culture was used to inoculate 900 mL of 2xYT medium

supplemented with ampicillin (100 mg/L) and chloroamphenicol (50 mg/L). The culture was grown at 30 °C and protein expression was induced during logarithmic growth (OD600 = 0.4-0.6) by addition of IPTG (Sigma-Aldrich) up to 1 mM. After 5 h of expression, the cells were harvested by centrifugation (4000 g at 4 °C for 15 min) and the pellet was stored at -20 °C overnight.

After thawing, the cell pellet was resuspended in 15 mL lysis buffer (50 mM NaH₂P0 , 10 mM imidazole and 1300 mM NaCI, pH 8.0) and incubated with lysozyme (1 mg/mL) for 30 min on ice. The cells were then lysed by ultrasonic disruption (5 times 5 s, 100% duty cycle and output control 3, Branson Sonifier 250, Marius Instruments Nieuwegein, the Netherlands). The lysate was centrifuged (13000 RPM at 4 °C for 15 min, Microcentrifuge, suspension was loaded onto a column and the flow-through was collected and the column was washed twice with 5 ml_ wash buffer (50 mM NaH₂P0₄, 20 mM imidazole and 1300 mM NaCI, pH 8.0). Finally, the CPs were eluted from the column using 10 times 1 ml_ elution buffer (50 mM NaH₂PO₄, 250 mM imidazole and 1300 mM NaCI, pH 8.0). The purification was analyzed by SDS-PAGE (Figure 2). The fractions containing the CPs were combined and dialyzed against a capsid buffer of pH 7.5 (50 mM Tris, 500 mM NaCI, 10 mM MgCI₂, 1 mM EDTA, pH 7.5) to obtain the protein dimers. For storage the proteins were assembled by dialysis against a pH 5.0 buffer (50 mM NaOAc, 500 mM NaCI, 10 mM MgCI₂, 1 mM EDTA, pH 5.0). The proteins were produced with a yield of 60-80 mg/L of culture and the purity was verified by SDS-PAGE. ESI-TOF: H₆-CP calculated 22506.6 Da, found 22507.4 Da and H₆-ELP[V₄L₄Gi-9]-CP(AN26) calculated 22253.4 Da, found 22253.4 Da (Figure 3).

Assembly experiments

For the pH-induced and salt-induced assembly of CPs, dialysis was used to change buffer composition. Therefore, 400 μΙ_ of CP-dimer solution (1.0 mg/mL in 50 mM Tris, 500 mM NaCI, 10 mM MgCI_2> 1 mM EDTA, pH 7.5) was inserted into a dialysis membrane (Spectra/Por 4 dialysis tubing, 12-14 kDa MWCO, 10 mm flat width) and dialyzed against 100 ml_ of the desired buffer. After 30 min the buffer solution was refreshed and the solution was incubated for another 30 min. All these steps were performed at room temperature (20 °C)

For temperature-induced assembly analyzed by SEC, a solution of CP-dimer (1.0 mg/mL in 50 mM Tris, 500 mM NaCI, 10 mM MgCI₂, 1 mM EDTA, pH 7.5) was inserted into the dialysis membrane and dialyzed against a pH 7.5 capsid buffer with an increased NaCI concentration (50 mM Tris, 1300 mM NaCI, 10 mM MgCI₂, 1 mM EDTA, pH 7.5) as described above.

Then the sample was incubated at the desired temperature (20 °C or 35 °C) for 15 min prior to injection onto the SEC-column. EDTA, pH 7.5) was inserted into the dialysis membrane and dialyzed against a pH 7.5 capsid buffer with an increased NaCI concentration (50 mM Tris, 500-2500 mM NaCI, 10 mM MgCI₂, 1 mM EDTA, pH 7.5) as described above. Then the protein solution was used to prepare TEM grids at 4 °C (cold room) and 20 °C (room temperature). For this purpose, the sample was diluted fivefold with the same buffer at the desired temperature (4 °C and 20 °C) and after the sample was incubated at that temperature for 30 min, it was applied to the TEM grid.

The DLS measurement of assembled H₆-ELP[V₄L G 9]-CP(AN26) is shown in Figure 4. Prior to the DLS measurement, the VLPs (abbreviation of "virus-like particles", i.e. VLPs are nanoparticles) were purified by SEC. Three measurements were performed for each sample.

Size exclusion chromatography (SEC)

The SEC measurements were performed on a Superose 6 10/300 GL column from GE Healthcare. For a typical analysis, 100 μί sample (100 g) was separated on the column with a flow rate of 0.5 mL/min. All measurements, except for the temperature-variation and multi-angle laser light scattering measurements, were performed at room temperature (20 °C) on an Amersham Ettan LC system (GE Healthcare, Diegem, Belgium) equipped with a fraction collector. For the temperature-variation

measurements, a Shimadzu LC-20A Prominence system (Shimadzu, 's Hertogenbosch, The Netherlands) was used. Here, the column, the injection loop and the buffer were positioned in the column oven and were equilibrated until the desired temperature was reached. The size exclusion

chromatography - multi angle laser light scattering (SEC-MALLS) experiments were conducted at room temperature using the Superose 6 0/300 GL column in line with a Wyatt DAWN HELEOS II light scattering detector using a laser operating at 658 nm and a Wyatt Optilab Rex refractive index detector. Overnight flushing of the system was performed to pre-equilibrate for every buffer followed by calibration using Bovine Serum Albumin. Weight-averaged NaCI, 10 mM MgCI₂, 1 mM EDTA, pH 7.5) and a dn/dc value of 0.185 for the pH 7.5, 0.5 M NaCI buffer (50 mM Tris, 500 mM NaCI, 10 mM MgCI₂, 1 mM EDTA, pH 7.5) and for the pH 5.0, 0.5 M NaCI buffer (50 mM NaOAc, 500 mM NaCI, 10 mM MgCI₂, 1 mM EDTA, pH 5.0).

Cryo-EM micrographs of assembled H₆-ELPrV₄UGi-9]-CP(AN26) are shown in Figure 5. These are selected images of the frozen-hydrated specimens of the pH-induced assemblies (A) and ELP-induced assemblies (B). The inserts show the translationally-aligned averaged images. Note that both VLPs appear to have the morphology of two concentric shells.

Figure 6 shows a size exclusion chromatogram for assembly in pH

7.5 buffer with varying NaCI concentrations. The assembly of H6-ELP[V₄L₄G 9]-CP(AN26) is analyzed in pH 7.5 buffer with 1.4 M NaCI (dashed line), 1.5 M NaCI (solid line) and 1.6 M NaCI (dotted line) at 20 °C.

Figure 7 shows a size exclusion chromatogram for assembly of control proteins and SDS-PAGE of the CPs. The assembly of H₆-ELP[V₄L₄Gr 9]-CP(AN26) (black line), H₆CP (dark grey line) and WT-CP (light grey line) in a pH 7.5 buffer with 1.8 M NaCI was analyzed. The SDS-PAGE depicts the CPs used in this study, from left to right: H₆-ELP[V₄L₄G₁-9]-CP(AN26), WT- CP and H₆CP.

Figure 8 shows uranyl acetate stained TEM micrographs of H₆-

ELP[V L₄Gi-9]-CP(AN26) prepared at different temperatures and NaCI concentration. The assembly was analyzed at pH 7.5, 2.0 M NaCI (A and B) and 2.3M NaCI (C and D) at 4 °C (A and C) and 20 °C (B and D). Cryo-electron microscopy (cryo-EM)

To prepare cryo-EM specimen, 4 μΙ aliquots of sample were applied on a glow-discharged holey carbon-coated grid (Quantifoil, R2/2), and blotted with filter papers from both sides for 4.5 s. Subsequently the grid was plunged into liquid ethane bath cooled by liquid nitrogen using an FEI VitrobotTM. The grid was quickly transferred into a Gatan 626DH cryo-holder (Gatan Inc., Oxford, UK) and examined in a 300-kV JEM-3200FS transmission electron 4k CCD camera (Gatan UltraScanTM 4000) and the yielding pixel size of 1.48 A at the specimen space.

Only images that exhibited minimum astigmatism, specimen drift, and charging were processed using AUTO3DEM (version 4.01.11) software packages.[X. Yan, R. S. Sinkovits, T. S. Baker, J. Struct. Biol. 2007, 157, 73- 82] Individual particles were semi-automatically extracted from the digital micrographs using e2boxer.pyprogra [G. Tang, L. Peng, P. R. Baldwin, D. S. Mann, W. Jiang, I. Rees, S. J. Ludtke, J. Struct. Biol. 2007, 157, 38-46]. The defocus level of each micrograph was estimated to be from 0.5 pm to 4.0 pm under focus by RobEM. The random model reconstruction method was employed to generate the initial low-resolution 3-D models for both VLPs [X. D. Yan, K. A. Dryden, J. H. Tang, T. S. Baker, J. Struct. Biol. 2007, 157, 211- 225]. The determination of the origins and orientations was started with the PPFT program and further refined by PO2R program. The final 3-D models for pH-induced (T=3) VLP and ELP-induced (7=1) VLPs were computed from 6910 and 8775 particles using P3DR, respectively. The estimated resolution for pH-induced VLPs was 10.9 A and for ELP-induced VLPs was 10.2 A using standard Fourier shell correlation at a 0.5 threshold. The 3_TD density maps were visualized and analyzed using Chimera [F. Pettersen, T. D. Goddard, C. C. Huang, G. S. Couch, D. M. Greenblatt, E. C. Meng, T. E. Ferrin, J Comput Chem 2004, 25, 1605-1612].

Claims

1. Protein comprising

- a first polypeptide segment capable of forming a nanocapsule by

- a second polypeptide segment capable of undergoing an inverse

temperature transition, the segment comprising two or more monomeric units selected from the group of tetrapeptides, pentapeptides and hexapeptides.

2. Protein according to claim 1 , wherein the two or more monomeric units are derived from elastin.

3. Protein according to claim 1 or 2, wherein the two or more monomeric units are pentapeptide monomeric units of Xaa-Pro-Yaa-Zaa-Gly, wherein, for each pentapeptide monomeric unit, Xaa is independently chosen from the amino acids isoleucine and valine, Yaa is independently chosen from the amino acids glycine and alanine and Zaa is independently chosen from the group of alpha-alkylated alpha amino acids and glycine.

4. Protein according to claim 3, wherein Xaa is valine and Yaa is glycine.

5. Protein according to any one of claims 1-4, wherein the number of

oligopeptide monomeric units is in the range of 2-160, preferably in the range of 5-50, more preferably 9.

6. Protein according to any one of claims 1-5, wherein the first polypeptide

segment is of viral origin.

7. Protein according to claim 6, wherein the first polypeptide segment is derived from the Cowpea Chlorotic Mottle Virus.

8. Protein according to claim 7, wherein the first polypeptide segment has the amino acid sequence of SEQ ID NO. 1.

9. Method for preparing a protein according to any one of claims 1-8, comprising - constructing a vector coding for bacterial expression of a polypeptide

capable of forming a nanocapsule by assembling with a plurality of other polypeptides that are also capable of forming the nanocapsule, then; comprising two or more monomeric units selected from the group of tetrapeptides, pentapeptides and hexapeptides, then;

expressing the protein, and then;

purifying the expressed protein.

10. Composition comprising nanocapsules and an aqueous solvent, the

nanocapsules comprising a plurality of proteins according to any one of claims 1-8.

11. Composition according to claim 10, wherein the second polypeptide segments of the plurality of proteins are at the interior of the nanocapsules.

12. Composition according to claim 10 or 11 , wherein the nanocapsules

encapsulate certain matter, such as a pharmaceutically active compound or an enzyme.

13. Method for preparing a composition according to any one of claims 10-12, comprising mixing the protein according to any one of claims 1-8 with an aqueous solvent, followed by incubating the resulting composition.

14. Method according to claim 13, wherein the aqueous solvent comprises

additional matter capable of being encapsulated by the nanocapsules that are formed in this method.

15. Use of a composition according to any one of claims 10-12 for the controlled release of encapsulated molecules or as template for the synthesis

nanoporous materials.