Field of invention

The field of the invention is new polypeptide material that is composed of elastin-like segments and coiled-coil segments and optionally functional polypeptide domain, which define application of the polypeptide material. The field of the invention is a protein of polypeptide material and DNA coding protein of the polypeptide material. The field of invention is also application of polypeptide material for promoting growth of cells, tissues or organs, inhibition of growth of pathogens, and for medical and pharmaceutical material used to treat living tissue, preferentially human or animal tissue.

State of the art

Various materials have been developed as biocompatible scaffolds for cell and tissue growth and represent an important issue not only for culturing cells in vitro but also for tissue repair and other tissue engineering and medical applications. Numerous synthetic polymers (for example polystyrenes, polyethylene vinyl acetates, polypropylenes, polyethylenes etc.) and biopolymers (including proteins such as collagen and fibrin and polysaccharides such as glycosaminoglycans and alginate) can provide a template for cell division, migration and differentiation.

Rationally designed peptides offer a promising way of constructing biomaterials with. For example, various cases of novel self-assembling and self-complementary amphiphilic peptides with characteristic alternating hydrophobic and hydrophilic amino acids have been reported. They form a variety of structures based on the formation of β-sheet fibrils among the amphiphiles (U.S. Pat. Appl. Pub. 0209145 Al) and can also be conjuncted with biologically active components and/or hydrophobic alkyl tails on termini (U.S. Pat. Appl. Pub. 0181973 Al, U.S. Pat. 7371719 B2).

In order to promote cellular ingrowth or function, scaffolds that are biocompatible and mimic the extracellular matrix (ECM) represent an advantageous environment for cell growth. Extracellular matrix (ECM) is composed of heterogeneous macromolecules including proteins and polysaccharides which form a three dimensional environment for cell growth representing a scaffold for stabilization and support of cell layers and tissues.

One of the components of ECM are rope-like elastic fibres, composed of amorphous protein elastin and microfibrils constructed primarily of fibrillin- 1. Precursor of stable, cross-linked elastin is a soluble molecule tropoelastin. This molecule consists of two types of domains: hydrophobic domains with high fraction of Gly (G), Val (V), Ala (A) and Pro (P) residues which often occur in repetitive sequences (first identified were pentapeptide VPGVG, hexapeptide VGVAPG and tetrapeptide VPGG) and hydrophilic domains with mainly Ala and Lys residues important in cross-linking which subsequently leads to formation of highly insoluble and extremely stable polymer.

Tropoelastin and elastin-related polymers are capable of coacervation between hydropobic domains previous to intermolecular covalent crosslinking, leading to self-assembly through temperature-driven phenomenon. This remarkable feature of intrinsic tendency for self- aggregation and significant stability of elastin and elastin-related polymers render elastin an ideal compound for the development of synthetic nanomaterials.

Polypeptides with elastin-like hydrophobic repetitive sequences were thoroughly analyzed and serve as soluble models of elastin (Lee et al., 2001, Biomacromolecules. 2, 170-179). But to employ these polypeptides as resilient biomaterials, it is necessary to cross-link the polypeptide chains. This can be for instance done by using γ-irradiation, chemical cross- linking or the cross-linking can be enzyme-mediated. Several elastin-like polypeptide materials have been prepared where a few modifications to hydrophobic repetitive sequences have been made including replacement of several amino acids by potential cross-linking residues or adding a cross-linking component (substrate for lysyl oxidase) to the elastomeric component (terra- or pentapeptide repeating units or mixture thereof) (U.S. Pat. 4589882). In order to obtain a better cross-linkable system, a material providing free amino groups on one intermediate of VPGVG peptide and free carboxyl groups on the other enabling cross-linking by chemical reagent has also been prepared (U.S. Pat. 4187852).

Exploiting recombinant technology, multimodular elastin-based polypeptides were constructed tuning the mechanical and functional properties of elastin-based materials. These include hybrids between elastin-like peptides and C5 domain of fibronectin promoting cell attachment (Welsh et al., 2000, Biomacromolecules. 1, 23-30) or hybrids between silk fibroin- like crystalline blocks and elastin-like blocks (Cappello et al., 1990, Biotechnology Progress. 6, 198-202). Elastin (-like) compounds are therefore promising in the field of nano- biomaterials (serving as substrates for cell growth, materials for drug or growth factor delivery) and tissue engineering (including skin substitutions, vascular grafts, heart valves and elastic cartilage).

Assembled material may also contain various additional functional biologically active segments and can for example, have an impact on cell attachment, cell adhesion, migration or proliferation. Tethering of functional moieties to the scaffolds represents an important feature when constructing self-assembling peptides. Using this principle targeting desired cell target and a decrease of the amount of used substance that must be employed in order to have a desired local effect could be achieved. Delivery of certain growth factors in soluble form can represent an additional drawback because cells loose responsiveness to the factor due to internalization and down-regulation of growth factor receptor.

The invention relates to the polypeptide material composed of elastin-like segments crosslinked by noncovalent interactions using a novel approach based on selected or designed coiled-coil segments therefore avoiding the need for the chemical modification of elastin-like repeats with introduction of noncovalent cross-linking sites. The invention is further improved by using regulated assembly and disassembly of the polypeptide material. Furthermore, presented polypeptide material can be additionally improved by added functional protein domains for cell growth promotion, differentiation, inhibition of microbial growth, binding metal ions, cytotoxicity, cell reprogramming or many other functions and therefore represents a novel biomaterial for cell/tissue/organ growth and treatment of living human or animal tissue. This approach allows almost endless number of combinations and furthermore allows the attachment of functional protein domains to the assembled biomaterial after the initial assembly, which may be used to design the time course of the therapy. Summary of the invention

The invention refers to a polypeptide material that contains at least one elastin-like segment and at least two coiled-coil segments, where at least one elastin-like segment is located between two or more coiled-coil segments and where separate polypeptide molecules are crosslinked by interactions between coiled-coil segments on those molecules. The invention refers also to the polypeptide material that optionally contains at least one functional polypeptide domain selected among, but not limited to: growth factors, cell differentiation factors, antimicrobial peptides, domains for metal binding and inhibitors of bacterial growth. Growth factors are selected preferentially from, but not limited to, epidermal growth factors (EGF), fibroblast nerve factors, nerve growth factors (NGF), preferentially NGF. The antimicrobial peptides are selected preferentially from, but not limited to, cathelicidins and defensins, preferentially cathelicidin LL-37. The domain for binding metal ions such as AEA or hexa histidine-tag, preferentially SEQ ID NO: 26. One or more functional domain can be either covalently linked to the polypeptide material or is introduced into the polypeptide material via interaction between coiled-coil segment fused to the functional domain and coiled-coil segments of the polypeptide material.

Invention also comprises polypeptide material composed of different ratios of polypeptide components, which can include different functional domains.

The polypeptide material according to invention contains at least one elastin-like segment at least two coiled-coil segments and optionally at least one functional polypeptide domain and elastin-like segment is of animal or human elastin, their mutants or synthetic elastin-like segment with preserved functional properties of elastin comprising at least 4 elastin-like repeats which include, but are not limited to, pentapeptide Val-Pro-Gly-Val-Gly or Gly-Val- Gly-Val-Pro or Gly-Val-Gly-Ile-Pro, additionally the elastin-like segment within this invention is defined to be composed of at least 12 amino-acid residues whereas the sum of the (% (n/n) of glycine residues) and double of the (% (n/n) of proline residues) should be higher than 60 % (n/n).

The invention refers also to the polypeptide material as described above, where the number of elastin-like segments is between 1 and 50, preferentially is between 2 and 10 number of coiled-coil segments is between 2 and 50, preferentially is between 3 and 10, and at least in one case the elastin-like segment is placed between coiled-coil segments.

The invention refers also to the polypeptide material as described above, where the coiled-coil segments are represented by naturally occurring or designed coiled-coil motifs comprising at least two heptads and can be either parallel or anti-parallel and are able to form either homooligomers with oligomerization state between 2 and 7 or heterooligomers with oligomerization state between 2 and 7.

The invention refers to the polypeptide material where the coiled-coil segments that crosslink different molecules are selected from the designed peptide pairs coiled-coil segments are preferentially selected among pairs SEQ ID: 10 and SEQ ID: 12, SEQ ID: 28 and SEQ ID: 30; SEQ ID:32 and SEQ ID: 34, SEQ ID: 36 and SEQ ID: 38 or SEQ ID: 14 or SEQ ID: 26.

The invention refers to a polypeptide material composed of at least two different polypeptide materials described above, where the composite polypeptide material is prepared by combining at least two polypeptide materials, and the coiled-coil segments of one polypeptide material can form heterooligomers with coiled-coil segments of the second material representing a means by which crosslinking of the composite polypeptide material can be regulated.

The invention refers to the peptides comprising coiled-coil segments that are in only one of the components of the polypeptide material described above and the said peptides are used for the disassembly of the polypeptide material which enables gentle cell recovery procedure.

The invention refers to the polypeptide material described above, where the segments and domains in above mentioned polypeptide material are optionally linked to each other with linker containing from one to 20 amino acids, preferentially from one to 6 amino acids, protein optionally contains a signaling sequence directing protein secretion and amino acid tag/tags.

The invention refers to DNA coding the protein described above and DNA is operatively linked to the regulatory elements, promoter and terminator to promote expression of the fusion protein in the host organism.

The invention refers to a process for preparation of polypeptide material described above composed of following steps: a) cultivating host organism expressing protein coded by DNA all described above; b) isolating expressed protein; and c) mixing purified protein/proteins to form polypeptide material as described above.

The invention refers to application of the polypeptide material described above for growth of cells, tissues or organs, optionally providing desired functional properties for cell growth. The invention also refers to application of the polypeptide material described above for the medicinal and pharmaceutical material used to treat living tissue, preferentially human or animal tissue. For example, but not limited to the replacement of an injured tissue, as prosthesis, for cell regeneration, cell reprogramming and as pharmaceutical material such as sheets for wound and burn healing, localized delivery of cytotoxic or cytostatic polypeptides.

The invention also refers to application of the polypeptide material described above for inhibiting growth of pathogens in case the functional polypeptide domain of the polypeptide material are antimicrobial peptides or domains for metal binding, especially SEQ ID NO: 26., which forms silver nanoparticles.

Figure legend

Figure 1 : Schematic review of the invention. A polypeptide material is composed of elastin- like segments and coiled-coil segments. Coiled-coil segments oligomerize and connect individual polypeptide chains of the polypeptide scaffold of the biomaterial. Functional polypeptide domains can be either incorporated into the polypeptide chain as part of the fusion protein or can be linked to the scaffold by interactions between coiled coil segments of the scaffold and coiled-coil fused to the functional polypeptide.

Figure 2: Antimicrobial potency of the polypeptide material which contains functional polypeptide domain, the antimicrobial peptide LL-37. The image contains the following samples: 1-MilliQ, 2-6 increasing concentrations of polypeptide material containing functional polypeptide domain comprising antimicrobial peptide LL-37; 2- 0,1 mg/ml, 3- 0,5 mg/ml, 4- 1 mg/ml, 5- 5 mg/ml, 6- 10 mg/ml.

Figure 3: PC 12 cell differentiation on the polypeptide material which contains functional polypeptide domain comprising growth factor NGF. The image contains: [A, B] HEK293T cells grown on polypeptide material [A] without any functional polypeptide domain, [B] containing functional polypeptide domain comprising growth factor NGF; [C, D] PC12 cells grown on polypeptide material [C] without any functional polypeptide domain, [D] containing functional polypeptide domain comprising growth factor NGF.

Figure 4: Antimicrobial potency of polypeptide material that contains functional polypeptide domain, AEA, that forms silver nanoparticles. Figure shows that bacterial growth is suppressed when functional polypeptide domain that forms silver nanoparticles is used.

Figure 5: Circular dichroism spectra of the representative pair of designed coiled-coil segments PI and P2, demonstrating the assembly of heterodimers.

A detailed description of the invention

Unless defined otherwise, all technical and scientific terms used herein possess the same meaning as it is commonly known to experts in the field of invention. The terminology to be used in the description of the invention has the purpose of description of a particular segment of the invention and has no intention of limiting the invention. All publications mentioned in the description of the invention are listed as references. In the description of the invention and in the claims, the description is in the singular form, but also includes the plural form, what is not specifically highlighted for ease of understanding.

Polypeptide material

The basis of the invention is a discovery that at least one elastin-like segment with at least two coiled-coil segments form a polypeptide material. The presented invention describes the polypeptide material whose composition is based on recent realization of inventors in the field of nano-materials. Invention is based on the discovery that a protein composed of at least one elastin-like segment and at least two coiled-coil segments form the polypeptide material that has specific physical properties, for example, to promote growth of cells, tissues or organs. Elastin-like segments mimic an elastin, provide elastomeric properties and are linked via oligomerization of coiled-coils which leads to formation of the polypeptide material. The polypeptide material therefore mimics extracellular matrix, is biocompatible, directs cellular growth and provides an appropriate environment for cell function. The term "polypeptide material" in the description of the invention refers to the material composed of proteins containing at least one elastin-like segment and at least two coiled-coil segments and its structure is two or three dimensional. Elastin-like segments provide elastomeric properties to the material while the coiled-coil segments enable the linking of elastin-like segments.

The inventors have come to the discovery that the elastin-like repeats can be linked into the polypeptide material by a novel approach using coiled-coil segments, which oligomerize. That is an improvement from previous discoveries, since the elastin-like repeats in order to be cross-linked had to be modified with the introduction of potential cross-linking sites (U.S. Pat. 4589882; U.S. Pat. 4187852) .

The term "elastin-like segment" in the description of the invention has a general meaning and refers to homologous segments of animal or human elastin composed of at least 4 elastin-like repeats. The elastin-like segments can be thread in continuous fashion or can be in at least one case placed between coiled-coil segments. The term "elastin-like segment" refers also to mutated or synthetic, artificially built, elastin-like segment that have preserved elastin-like characteristics. Elastin-like segments could be chemically synthesized or recombinantly expressed. The term "homologous segments" in the description of the invention refers to the amino acid sequences of protein, originating from the same or another organism, which show a good protein alignment, preferentially more than 50 % conserved structure, preferentially 60 %, preferentially 70 % in the alignment analysis. The term "homologous segments" refers also to mutant protein segments, whose mutations minimally alter the amino acid sequence.

The term "elastin-like repeats" also refers to hydrophobic repetitive sequences present in elastin, which contain many Gly (G), Val (V), Ala (A) and Pro (P) residues often occurring in tandem repeats of several (from 3 to 6) amino acids. The term elastin-like repeats includes, but is not limited to, pentapeptide sequences Val-Pro-Gly-Val-Gly or Gly-Val-Gly-Val-Pro or Gly-Val-Gly-Ile-Pro.

The term "elastin-like repeats" also refers to sequences composed of at least 12 amino acid residues and where the sum of the (% (n/n) of glycine residues) and 2* (% (n/n) of proline residues) should be higher than 60 % (n/n). Namely, the onset of elastomeric function of elastin is controlled by just two amino acid residue type, glycine and proline. There is a combined proline and glycine threshold above which the elastomeric properties of elastin are apparent. Another feature of elastomeric domains having proline and glycine content above threshold, is that they are devoid of a-helix and β-sheet, but instead remain in disordered form, also when aggregated (Rauscher et al. 2006, Structure. 14, 1667-1676).

The basis of the invention is also the discovery that the number of elastin-like segments is between 1 and 50, preferentially is between 2 and 10, and number of coiled-coil segments is between 2 and 50, preferentially is between 3 and 10, and at least in one case the elastin segment is placed between coiled-coil segments and the coiled-coil segments are able to form homooligomers or heterooligomers with oligomerization state between 2 and 7.

The term "homooligomerization" in the description of invention has a general meaning and refers to a process of forming complexes composed of only one type of monomers wherein the number of monomers is between 2 and 7.

The term "heterooligomerization" in the description of invention has a general meaning and refers to a process of forming complexes composed of different types of monomers wherein the number of monomers is between 2 and 7.

The term "monomer" in the above description refers to one coiled-coil segment that may interact with the other coiled-coil segments in such manner that they twist around each other.

The term "coiled-coil segment" has a general meaning and refers to structural protein motives comprising 2 or more a-helices that twist around each other forming a super coil. They contain a heptad repeat designated (a-b-c-d-e-f-g) _n every two turns of a helix, "a" and "d" usually represent nonpolar, hydrophobic residues that are found at the interface of the two helices, "e" and "g" are solvent exposed polar residues that interact electrostatically, "b", "c" and "Γ are hydrophilic and exposed to the solvent. Different amino acids on positions "a-g" define oligomerization state, specify, helix orientation and stability. More specifically, the term coiled-coil segment in the description refers to naturally occurring or designed coiled- coil protein structure motives which comprise at least two heptads and can be parallel or antiparallel and can form homo- or hetero-oligomers.

The coiled coil segments could be selected from naturally occurring or designed coiled-coil protein structure motives, preferentially from naturally occurring leucine-zipper proteins such as BZIP or designed coiled-coil sequences, preferentially from SEQ ID: 14 or SEQ ID: 26 or pairs SEQ ID: 10 and SEQ ID: 12, SEQ ID: 28 and SEQ ID: 30; SEQ ID:32 and SEQ ID: 34, SEQ ID: 36 and SEQ ID: 38.

Functional polypeptide domain

The invention refers to the discovery that the polypeptide material can have in addition to the structural role of coiled-coil and elastin-like domains incorporated at least one additional functional polypeptide domain, which gives the polypeptide material additional functional property such as promoting the growth of the cells and cell differentiation, inhibiting growth of pathogens or binding metal ions.

The functional polypeptide domain could be covalently linked to at least one elastin-like segment with at least two coiled-coil segments of polypeptide material.

Besides linking functional domain covalently to the polypeptide material as described above, the invention refers also to another manner of incorporating the functional polypeptide domain into the polypeptide material. The inventors have come to the discovery that it is beneficial to provide some functional properties to the polypeptide material that does not contain any functional polypeptide domain. The inventors have found that the functional polypeptide domain fused with coiled-coil segments can be added to the polypeptide material and these functional polypeptide domain fused with coiled-coil segments become incorporated into the polypeptide material via linking by oligomerization with complementary coiled-coils present in the polypeptide material.

By this, the polypeptide material already containing some functional polypeptide domain, can gain additional desired properties via adding other functional polypeptide domain fused with coiled-coil segments complementary with coiled-coil segments of the polypeptide material. Functional polypeptide domains can be added to the polypeptide material at the time of its initial assembly or subsequently and additionally it can be added to the organism at different location, while it can be targeted to the site of the implanted polypeptide material with complementary coiled coil segments.

When the functional polypeptide domain is covalently linked to the polypeptide material, it is available for cell, tissue, organ. Local concentration of the functional polypeptide domain is higher due to the absence of diffusion in contrast to soluble functional polypeptide domain. Also, less functional polypeptide domain is needed in order to have the desired local effect as if the domain would be in soluble form. Additionally its potential toxicity and harmful effects are localized to the site of implanted polypeptide material. This type of material also allows potential different time course of therapy by subsequent delivery of different functional proteins.

The term "fused or fusion" has a general meaning and in the description refers to a process of making a hybrid protein from two previously separate proteins/domains/segments, optionally linked to each other with linker containing from one to 20 amino acids.

The term "covalently linked" has a general meaning and refers to the functional polypeptide domain being bound via peptide bond to the polypeptide material.

The term "complementary" refers to the ability of a coiled-coil segment of homo- or hetero- oligomerization with the other coiled-coil segment.

The term "functional polypeptide domain" refers to the molecules providing the material some functional properties such as cell growth promotion, inhibition of bacterial growth, cell differentiation or binding metal ions. The functional polypeptide domain is selected among, but not limited to, growth factors, antimicrobial peptides and domains for metal ion binding. -

The term "growth factor" has a general meaning and refers to the molecule that binds to the cell receptors and regulates the growth, replication or differentiation of target cells or tissues. Examples of growth factor include, but are not limited to, epidermal growth factor (EGF), fibroblast growth factor, nerve growth factor (NGF), erythropoietin and others; preferentially NGF; preferentially SEQ ID NO:. 24

The term "antimicrobial peptide" refers to the broad spectrum antibiotic which acts bactericidal, but can also act on fungi or viruses by disrupting membranes, interfering with metabolism, or targeting cytoplasmic components. Examples of antimicrobial peptides include, but are not limited to cathelicidins and defensins; preferentially cathelicidin LL-37; preferentially SEQ ID NO: 16.

The term "domains for metal binding" has a general meaning and refers to peptides which can bind to metals including, but not limited to, Ag, Au, Pd, Pt, which has an antimicrobial effect. The domain for metal binding is preferentially SEQ ID NO: 26, which is composed of 3 segments, capable of trimerization, and forms silver nanoparticles. The invention also encompasses the recognition that polypeptide material that also contains functional polypeptide domain covalently linked to the polypeptide material is a mixture of (i) polypeptide material that contains at least one elastin-like segment and at least two coiled coil segments with the (ii) polypeptide material that besides at least one elastin-like segment and at least two coiled-coil segments contains also a functional polypeptide domain.

Depending on the desired properties of the polypeptide material, the mixture can be composed of various ratios wherein the polypeptide material that besides at least one elastin-like segment and at least two coiled coil segments also contains a functional domain represents less than 50 % of a mixture, more typically 20 % and most typically 1-5 % of the mixture. The polypeptide materials can be mixed on molar, weight, volume basis etc.

Such heterogenous mixtures can have certain advantages over the homogenous mixtures. Namely, the homogenous mixtures comprising only polypeptide material that contains at least one elastin-like segment and at least two coiled coil segments do not provide the desired properties to cells such as enabling cell growth promotion, cell adhesion, inhibition of bacterial growth, cell differentiation, metal ion binding. Whereas on the other side, the homogenous mixtures comprising only polypeptide material that besides at least one elastin- like segment and at least two coiled coil segments contains also a functional domain could form the polypeptide material which is weaker in comparison with the polypeptide material that contains at least one elastin-like segment and at least two coiled-coil segments.

In order to combine the desired features regarding polypeptide materials composed of different belonging functional domains the invention also encompasses mixing multiple polypeptide materials with different belonging functional parts with the polypeptide materials without functional part.

The polypeptide material that is disassembled using peptide comprising coiled-coil segment

The basis of the invention is also the discovery that the polypeptide material can be disassembled by the addition of peptide comprising coiled-coils segments that are also present in the polypeptide material.

The inventors have discovered that the addition of the peptide comprising coiled-coil segment similar to coiled-coil segment of the polypeptide material represents a loose approach by which the disassembly of the polypeptide material can be achieved. The added coiled-coil segment interferes with structure of the polypeptide material due to its ability of oligomerization with one of the coiled-coil segments of the polypeptide material, which can loosen the polypeptide material and lead to its disassembly.

In such a manner, cells growing on the polypeptide material are recovered without chemical changes (e.g. pH, ionic strength), physical changes (e.g. long incubation periods at high temperatures, osmotic shock) or enzymatic digestion of the polypeptide material that can affect or damage surface receptors and adhesion molecules or stress cells. Recovered cells can be counted, replated or used for subsequent biochemical analysis or technological applications.

The term "peptide comprising coiled-coil segment" in the description refers to coiled-coil segment wherein this segment is capable of oligomerization with one of the coiled-coil segments of polypeptide material. The term "coiled coil segment" refers to coiled-coil segments described above.

Polypeptide material composed from at least two different polypeptide materials

The basis of the invention is also the discovery of the polypeptide material that is a combination of at least two different polypeptide materials. The composite polypeptide material is prepared by combining two polypeptide materials where the coiled-coil segments of one material can form heterooligomers with coiled-coil segments of the second material. The inventors have come to the discovery that heterooligomerization of the coiled-coil segments from one material with the coiled-coil segments of the second material can represent an approach by which an initiation of the polypeptide material formation can be regulated. Namely, one soluble polypeptide material containing a coiled-coil segment capable of heterooligomerization does not assemble if it presents the only material in the solution. The formation of polypeptide material can be initiated only when the second material is added which contains a coiled-coil segment, which heterooligomerizes with the coiled-coil segment of the first material and thus represents a mechanism of regulating assembly of the material.

The composite material contains at least two polypeptide materials but could be composed of several, up to ten, preferentially between two and eight different polypeptide materials according to the invention. Protein of polypeptide material

The invention refers to a protein of the polypeptide material described above. Protein contains at least one elastin-like segment and at least two coiled-coil segments, where at least one elastin-like segment is located between two or more coiled-coil segments and where separate polypeptide molecules are cross-linked by interactions between coiled-coil segments on those molecules Additionally, protein of the polypeptide material could contain functional polypeptide domain, which could therefore optionally be a part of the protein. The functional polypeptide domain as being part of the protein of the polypeptide material is covalently linked and could not diffuse away, which represents a certain advantage.

The invention refers to the protein, which contains fused coiled-coil segments with functional polypeptide domain. Inventors have come to the discovery that functional polypeptide domain can become incorporated into the polypeptide material via interactions of their coiled-coil segments complementary with coiled-coil segments of the material. By this means, thematerial can gain functional properties.

The segments and domains of the protein described above are optionally separated from each .other with linker containing from one to 20 amino acids, preferentially from one to 6 amino acids, proteins optionally contain signal sequence directing protein secretion and amino acid tags for purification and labelling.

The term "linker" in the description refers to shorter amino acid sequences, whose role could be only to separate the individual domains, segments of the protein. The role of the linker peptide in the protein, inclusion of which is optional, may also be the introduction of the splitting site or for posttranslational modifications, including the introduction of sites for improved processing. The length of the linker peptide is not restricted; however, it is usually up to 30 amino acids long, preferentially from one to 20 amino acids, more preferentially from one to six amino acids. Any amino acid could be included in a linker, preferentially amino acids are selected, but not limited to, serine, glycine, threonine, proline, valine, alanine residues that allow flexibility and specific intermolecular association only through coiled-coil or elastin-like domains..

The term "signal sequence" in the description refers to the amino acid sequence, which is important for directing the protein to a certain location in the cell. Signal sequences also vary depending on the host organism in which the fusion protein is expressed. Amino acid sequences of the signal sequences are well known to experts, as well as which signal sequence is functional in a certain organism.

The term "amino acid tag/tags" refers to the sequences of amino acids, which are added to the protein to facilitate purification, isolation or detection of the protein.

The position of the signalling sequence, linkers and amino acid tags is optional but it should allow functional expression of the protein and maintain the function for which these amino acid sequences were selected.

DNA coding protein of polypeptide material

The present invention relates also to DNA coding protein of the polypeptide material which contains at least one elastin-like segment and at least two coiled-coil segments, where at least one elastin-like segment is located between two or more coiled-coil segments and where separate polypeptide molecules are crosslinked by interactions between coiled-coil segments , on those molecules optionally linked with protein functional domain and the segments and domains are optionally linked to each other with linker. The protein optionally contains signaling sequence and amino acid tags. The present invention also relates to DNA coding protein of polypeptide material which contains fused coiled-coil segments with functional polypeptide domain and the segments and domains are optionally linked to each other with linker, protein optionally also contains signaling sequence and amino acid tags.

The term "DNA" in the description refers to a sequence of nucleotides having an open reading frame that encodes a protein of the subject invention, being operatively linked to the regulatory elements, promoter and terminator to promote expression of the protein in the host cells. The length of the DNA sequence may vary greatly depending on the particular protein.

The term "regulatory elements" in the description has a general meaning and refers to the region of DNA in expression vector where regulatory proteins such as transcription factors bind and control gene expression.

The term "promoter" in the description has a general meaning and refers to a region of DNA in expression vector facilitating the transcription of target gene. The type of the promoter depends on host organism in which the gene will be expressed. In case of expression in prokaryotes, namely E. coli, several promoters such as Pi _ac , P _TS and preferentially P _T7 can be used. In case of expression in eukaryotes, namely in mammalian cells, the promoters are in most cases of viral origin such as P _CMV or Psv40-

The term "terminator" in the description has a general meaning and refers to the sequence that marks the end of a gene for transcription. The type of the terminator depends on host organism in which the gene will be expressed. In case of expression in prokaryotes, namely E. coli, a terminator from bacteriophage T7 can be used.

Recombinant nucleic acid

Standard methods of molecular biology were used in the invention and are generally known to experts in the field (see Sambrook et al. 1989. Molecular Cloning: A laboratory manual, 2nd ed., Cold Spring Harbor, NY, Ausubel et al. Current Protocols in Molecular Biology, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., NY).

The invented proteins of the polypeptide material can be synthesized by expressing DNA coding for proteins, in a suitable host organism. The DNA coding for the proteins is inserted in an appropriate expression vector. Suitable vectors include, but are not limited to: plasmids, viral vectors, etc. Expression vectors, which are compatible with the host organism cells, are well known to experts in the field and include the appropriate control elements for transcription and translation of nucleic acid sequence. Typically, an expression vector includes an expression cassette, which includes in 5' to 3' direction the promoter, the coding sequence for the fusion protein operatively linked with the regulatory elements, promoter and terminator, including a stop codon for RNA polymerase and polyadenylation signal for polyadenylase.

Expression vector may be prepared for expression in prokaryotic and eukaryotic cells. For example, prokaryotic cells are bacteria, primarily Escherichia coli. According to the invention, prokaryotic cells are used to get a sufficient quantity of nucleic acid. Expression vector generally contains the operationally associated control elements which are operationally linked to the DNA of the invention, which codes for the protein. The control elements are selected in a way to trigger efficient and tissue-specific expression. The promoter may be constitutive or inducible, depending on the desired pattern of expression. The promoter may be of native or foreign origin (not represented in the cells, where it is used), and may be natural or synthetic. The promoter must be chosen in order to work in the target cells of the host organism. In addition, initiation signals for the efficient translation of fusion protein are included, which comprises the ATG and the corresponding sequences. When the vector, used in the invention, includes two or more reading frames, should the reading frames be operationally associated with control elements independently and the control elements should be the same or different, depending on the desired production of proteins.

Examples of bacterial expression vectors include, but are not limited to: pET vectors, pRSET vectors, and others. When vectors are used in the bacterial cells, the control elements are of bacterial origin.

Examples of mammalian expression vectors for mammalian cells include, but are not limited to: pcDNA (Invitrogen), pFLAG (Sigma), and others. When vectors are used in mammalian cells, the control elements are in most cases of viral origin, for example, adenovirus 2, cytomegalovirus, a virus Simian virus 40.

Host organism

The present invention also relates to a host organism expressing the above described proteins. The term "host organism" refers to the organism, in which the DNA coding for protein has been introduced in order to be expressed. The transfer of vectors into host organisms is carried out by conventional methods, known to the experts from the field, and the methods refer to transformation, transfection, including: chemical transfer, electroporation, microinjection, DNA lipofection, cell sonication, particle bombardment, viral DNA transfer, and more. In the context of the invention, the introduction of DNA is by transformation in to bacterial cells.

The host organism may be prokaryotic or eukaryotic. Eukaryotic cells, suitable for the fusion protein expression, are not limited as long as cell lines are compatible with propagation methods of the expression vector and with the expression of the fusion protein. The preferred eukaryotic cells include, but are not limited to, yeast, fungi, plant cells and mammalian cells, such as: mouse, rat, monkey or human fibroblasts.

For the DNA expression any bacterial host can be used according to the invention. For the expression of proteins of the present invention, the use of bacteria or yeast is preferred. The protein can be expressed in bacteria E. coli or B. subtilis or in yeast S. cerevisiae or P. pastoris. The preferred bacterium is E. coli. Invention refers to the expression of the protein in bacteria or yeast. Invention refers to bacteria or yeast, that express protein, preferably to the bacteria E. coil or to yeast P. pastoris.

Protein production/Process for preparation of polypeptide material

Invention also refers to a process of generating the polypeptide material and includes introducing DNA coding protein of polypeptide material into the host organism, cultivating host organism under conditions suitable for protein expression, isolation of protein and exposing protein to specific conditions to form polypeptide material.

According to the invention fusion protein can be synthesized in the host organism that expresses the heterologous nucleic acid, which encodes for the fusion protein. Invented fusion protein is used for preparation of polypeptide material. Optionally, a fusion protein is operatively linked to the signalling sequence that is coded in the nucleic acid.

In general, the heterologous nucleic acid is incorporated into an expression vector (viral or non- viral), which is described above.

The invention includes host cells and organisms that contain nucleic acid according to the invention (transient or stable), which codes for the fusion protein according to the invention. Appropriate host organisms are known to the experts in the field of molecular biology and include bacterial and eukaryotic cells.

The transfer of vectors into host cells is carried out by conventional methods, known in the state of the art, and described above.

The DNA transfer may be transient or stable. Transient expression refers to the introduction of the vector DNA, which according to the invention is not incorporated into the genome of cells. Stable intake is achieved by incorporating DNA of the invention into the host genome. The transfer of the DNA according to the invention, especially for the preparation of the host organism, which has a stable DNA integrated, may according to the invention be controlled by the presence of markers. DNA coding for markers refers to resistance to drugs, for example antibiotics, and may be included in the vector with the DNA according to the invention, or on a separate vector. The protein is expressed in the suitable host organism. Expression of large amounts of fusion proteins can be achieved in bacteria such as E. coli, in yeast P. pastoris, however it is also possible to use mammalian cell culture to produce smaller amounts of proteins when posttranslational modifications are necessary for correct folding and function. It is known that the protein can be expressed in mammalian cells of the following organisms: human, rodents, cattle, pig, poultry, rabbits and alike. The host cells can be cultivated primary cell lines or immortalized cell lines. Expression of fusion proteins can be constitutive or inducible, e.g. expression of protein, can be induced by addition of inducer IPTG, which by binding lac repressor turns on expression of protein of interest. Fusion proteins can be expressed either in soluble fraction or inclusion bodies.

Protein purification techniques including size-exclusion chromatography, ion-exchange chromatography, reverse-phase chromatography, affinity chromatography when fusion proteins or appended tags interact with purification column specifically etc. are well know to the experts in the field.

The assembly of the polypeptide material can be reached under appropriate conditions enabling the linking of elastin-like segments through coiled-coil segments. When polypeptide material is formed from single protein, appropriate conditions are found by screenings of solubility and secondary protein structure of soluble part but especially of precipitate in buffers of various pH (usually from pH 3 to pH 9), ionic strength, organic solvents (e.g. DMSO, acetonitrile, trifluoroethanol). The important factors for self-assembly are also the concentration of fusion protein and temperature. The polypeptide material is formed when concentration of protein is between 0.1 mg/mL and 20 mg/mL, typically from 0.5 to 10 mg/mL. Self-assembly of polypeptide material could also be triggered by heating the fusion protein precipitate over the first melting temperature and by further slow cooling down.

When the polypeptide material is designed to assemble from several fusion proteins, the self- assembly usually takes place after mixing the proteins in appropriate molar ratio in conditions favouring self-assembly.

The protein of the polypeptide material containing only at least one elastin-like segment and at least two coiled-coil segments, can be mixed in various ratios with the protein that also contains functional polypeptide domain. Depending on the desired properties of the polypeptide material, the mixture can be composed of various ratios wherein the protein that besides at least one elastin-like segment and at least two coiled-coil segments also contains a protein functional domain that represents less than 50% of a mixture, more tipically 20% and most tipically 1-5 % of the mixture. By mixing two proteins where the coiled-coil segments of one protein can form heterooligomers with the coiled-coil segments of the second protein, an initiation of polypeptide material formation can be regulated.

Protein containing fused functional polypeptide domain with coiled-coil complementary with coiled-coil segments of polypeptide material, can be mixed with the polypeptide material.

Through incorporation of functional parts fused with coiled-coil segments into the material, the later can gain functional properties. By adding peptides comprising coiled-coil segments that are in one of the two materials composed of polypeptide material, the disassembly of the polypeptide material leading to cell recovery can be achieved.

Application of the polypeptide material

The invention is also referring to the application of the polypeptide material. The basis of this is that polypeptide material can be used as material/pharmaceutical formulation for stimulating growth of cells, tissues or organs, cell differentiation, cell repair, cell reprogramming, cytotoxicity for medical and pharmaceutical material for living human or animal tissue, for preventing growth of pathogens.

The term "growth" has a general meaning and in the description refers to cell/tissue/organ development and cell division. In accordance with the present invention any cell type including, but not limited to, smooth muscle cells, fibroblasts, keratinocytes, epithelial, immune cells and endothelial cells can be cultured. Polypeptide material closely mimics the natural cell environment- extracellular matrix due to the presence of a network of elastin-like segments. Cells, tissues and organs can be cultured on the surface of polypeptide material and cells can extend processes into the structure or migrate into it in case the material forms three- dimensional structure.

Cells, tissues and organs should be cultured on polypeptide material close to physiological conditions in a similar manner to which they are cultured on convenient cell or tissue culture dish or slide. Depending upon the proliferation rate of cells, cell number and density, which vary depending upon the particular type of cells and purposes for which they will be used, cells may be cultured on polypeptide material for any appropriate time.

In case cells are cultured on the polypeptide material that additionally contains also at least one or combination of several, typically from 2 to 10 functional polypeptide domains selected among, but not limited to, growth factors, cell adhesion molecules, chemotactic molecules, receptor ligands, antimicrobial peptides, factors for cell reprogramming, factors for cell differentiation, cytotoxic or cytostatic polypeptides such as epidermal growth factors (EGF), fibroblast nerve factors, nerve growth factors (NGF), preferentially SEQ ID NO: 24, antimicrobial peptides such as cathelicidins and defensins, preferentially cathelicidin LL-37, preferentially SEQ ID NO: 16 .

Functional polypeptide domain therefore do not need to be added to the medium, but are instead bound to the polypeptide material on which cells grow which represents an important advantage. As already mentioned, it is also an object of the present invention to mix polypeptide materials with different functional polypeptide domain in optional ratios which can be the means of providing the desired properties for optimal cell growth and can be prepared in almost endless number of combinations.

Addition of peptides comprising coiled-coil segments that are incorporated in the polypeptide material to the said material can lead to its disassembly, which represents a gentle manner by which cells can be recovered and then counted, replated or used for subsequent biochemical analysis or different technological applications. In contrast to convenient methods including biochemical, physical changes or enzymatic digestion, the mentioned approach does not affect or damage cell surface molecules or represent a stress to the cells.

The basis of the invention is also the discovery that the polypeptide material can be used for medical and pharmaceutical material for treatment of living tissue, preferentially human or animal tissue.

Polypeptide material either with or without cells or tissues growing on the surface can be used as material/pharmaceutical composition for treatment of a variety of tissue defects or diseases and can be implanted into the body surgically or by other type of suitable procedure.

The term "treatment" has a general meaning and in the description refers to the procedure of remediation of damaged or injured human or animal tissue using polypeptide material. The term "preparation of medical and pharmaceutical material" refers to the procedures employed in order to arrange the polypeptide material in such shape or condition that it can be used e.g. for replacement of an injured tissue, as prosthesis and as pharmaceutical material such as sheets for wound and burn healing.

Polypeptide material of the present invention elicits minimal or no detectable immune or inflammatory response. In general, polypeptide material of the invention may be used in any situation involving injury or tissue damage resulting from surgery, trauma, tumor, degenerative disease or other diseases or conditions. Polypeptide material can be used for repair of natural elastic systems, especially those in which tropoelastin or elastin is naturally present, by replacing a damaged portion of the system such as a ligament, tendon, blood vessel wall, etc. with the polypeptide material. The polypeptide material can also be surgically inserted into a human or animal in place of diseased or missing vascular material and be as such used as a vascular replacement or patch. The polypeptide material is useful to restore or to aid restoring structural and/or functional integrity to the tissue.

The cells that have proliferated/differentiated on polypeptide material can be removed or extracted from the structure and further cultured in vitro in a culture vessel. Subsequently, they can be administered to a subject and may be used to supplement the tissue or organ.

The application of polypeptide material, which also contains functional polypeptide domain, e.g. growth factors, for treatment of living human or animal tissue represents certain benefit over treatment where growth factors would be used in soluble form. The soluble growth factors can namely have some undesired effects especially in vivo like stimulation of the growth of competing cells which overgrow the target cell or diffuse into the blood stream exerting their effects elsewhere. By adding functional polypeptide domain to the polypeptide material, the local effect of the functional domain to the target cell can be achieved. This invention also provides a means to inhibit growth of undesired tissue by localizing the cytotoxic or cytostatic effects to the site of application, particularly, but not limiting the effect on cancerous tissue.

When epithelial cells are seeded with an intention of reconstituting a piece of epidermis, polypeptide material can act as a skin base for transplantation to the receiver. The polypeptide material can also be used for construction of human elastin-like prostheses, e.g. tubes for blood vessel replacement and sheets for wound and burn healing. The prosthesis can become a permanent tissue replacement while the polypeptide material is subject to infiltration of patient cells, including endothelial cells. The polypeptide material can also be used to coat the surface of any type of prosthesis, including a prosthesis comprising a synthetic material, an animal material and/or a metal.

The term "prosthesis" refers to any material implanted into the body, including for blood vessel replacement, for heart valve replacement or cloth-like material. It can be also used as sheets for burns or wounds to promote healing.

Inventors have also come to the discovery that the polypeptide material when containing at least one elastin-like segment and at least two coiled-coil segments and additionally containing functional polypeptide domain in case represented by antimicrobial peptides such as cathelicidins and defensins, preferentially cathelicidin LL-37, preferentially SEQ ID NO: 16. and domains for metal binding, preferentially SEQ ID NO: 26, which forms silver nanoparticles, can be used for preventing growth of pathogens. The advantage of antimicrobial peptides or metals, particularly silver, is their broad-spectrum antimicrobial action.

The term "pathogens" in this description refers to common bacteria, fungi, yeast and viruses that can cause disease. Preferentially it refers to common bacteria which can cause disease such as, but not limited to, gram-positive bacteria S. aureus, S. pyogenes, S. pneumoniae, gram-negative bacteria H. influenzae, K. pneumoniae, L. pneumophila, P. aeruginosa, E. coli.

Infections often result as a consequence of any intervention into tissue. Considering the discovery that the polypeptide material can be used for replacement of a damaged or injured natural elastic tissue and be transplanted to the receiver, it is beneficial that the polypeptide material also prevents growth of pathogens and therefore extenuates or prevents the potential infection. Furthermore, the polypeptide material finds its use also in constructing elastin-like prostheses, e.g. tubes for blood vessel replacement and sheets for other uses such as wound and burn healing as already mentioned or can be used to coat the surface of the prosthesis. The prostheses can commonly present a threat to the patients due to biofilm formation potentially leading to many life-threatening infections, including infections caused for example by P. aeruginosa and S. aureus, two bacteria often found in biofilms and also infecting burn wound. In this view, the present invention presents the solution of extenuating or preventing infections, caused by pathogens colonizing prostheses and burn wounds.

Considering silver being nonselective and extremely active in small quantities, the authors have come to the discovery that polypeptide material containing domain for metal binding, preferentially SEQ ID NO: 26, which forms silver nanoparticles, represents a particularly promising material for preventing pathogen growth especially useful for wound dressings and prostheses.

Examples of implementation, designed to illustrate the invention, are shown below. The descriptions of examples of implementation have no intention of limiting the invention and should be understood as a demonstration of the invention.

Examples of implementation Example 1: DNA construct preparation.

Polypeptide material according to this invention was designed by combining elastin-like segments, coiled-coil segments and functional domains. Amino acid sequences of the heterodimeric parallel coiled-coil segments (SEQ ID: 10, SEQ ID: 12, SEQ ID: 28, SEQ ID: 30; SEQ ID:32, SEQ ID: 34, SEQ ID: 36, SEQ ID: 38) were designed based on the knowledge on the assembly of coiled-coil segments known to people skilled in the subject in order maximize the specificity of coiled-coil segment interactions. Amino acid sequence of the homodimeric parallel coiled coil segment was selected from the GCN4 peptide (SEQ ID: 14). Trimerization coiled coil sequence AEA (SEQ ID: 26) that forms silver nanoparticles was designed based on the knowledge on the assembly of coiled-coil segments known to people expert in the subject by including a repeat of three segments that are combined in a staggered heterotrimer and include at positions b, c and / of the coiled coil heptad residues Ala, Ala and Glu, respectively.

Elastin-like segments were selected from the repeats of the pentapeptide (SEQ ID: 18).

Functional domains were selected for this example among the nerve growth factor (SEQ ID: 24), antimicrobial peptides LL-37 (SEQ ID: 16), which were selected from the natural sequences of murine and human proteins. The fusion proteins may contain a signal sequence for localization of the protein and a peptide tag for detection or purification of protein.

DNA sequences optimized for protein expression in desired hosts (E.coli) were designed using amino acid sequences and tool programme Gene Designer from DNA2.0 Inc. version 1.0.0.1 (DNA2.0 Headquarter 1430 O'Brien Drive, Suite E, Menlo Park, CA 94025, USA). Genes were ordered from MR.Gene GmbH (Im Gewerbepark B32, D-93059 Regensburg) and cut out with restriction endonucleases. Only LL-37 DNA sequence was taken from a plasmid with ORF clone of Homo sapiens cathelicidin antimicrobial peptide (CAMP). Part of the gene coding LL-37 was amplified with PCR.

DNA constructs and corresponding fusion proteins are described in Table 1. Detailed description and function of individual DNA or protein product are described in Table 2. All DNA constructs have start codon (ATG) before tag of histidines. The constructs were cloned into pET-31b(+) vector for high-level expression of peptide sequences fused with the ketosteroid isomerase protein. The expression cassette includes in 5' to 3' direction the T7 promoter, the KSI coding and multiple cloning site for fusion protein, and T7 terminator. These regulatory elements enable expression of protein in prokaryotic cell line E.coli carrying T7 RNA polymerase.

DNA constructs were prepared by using methods of molecular biology that are basically described in molecular biology handbook (Sambrook J., Fritsch E.F., Maniatis T. 1989. Molecular cloning: A laboratory manual. 2nd ed. New York, Cold Spring Harbor Laboratory Press: 1659 str.). Plasmids, constructs and intermediate constructs were transformed with chemical transformation into bacterium E. coli DH5a or BL21 (DE3) pLysS.

Fusion proteins corresponding to designed DNA constructs are generally composed of at least one elastin-like segments and at least two coiled-coil segments optionally they contain functional polypeptide domain (e.g. NGF and/or LL-37). KSI-DP domain enables expression of proteins in inclusion bodies and acid cleavage at aspartate-proline (DP) linkages. Table 1 : Fusion proteins, which were used for the demonstration of the invention

Nucleotide/

plasmid o. Name Construct composition amino-acid

backbone sequence

KSI-DP-His _tag -LL37- DP-His _ta -LL37-ELST-GCN- SEQ ID NO: 1/

pET-31b(+)

ELST-GCN-ELST ELST -T7 _t SEQ ID NO: 2

KSI-DP-His _tag -LL37-

DP-His _ta -LL37-ELST-GCN- SEQ ID NO: 3/

ELST-GCN-P2-ELST- pET-31b(+)

P2-ELST-NGF -T7, SEQ ID NO: 4

NGF

SEQ ID NO: 9/

PI Synthetic

SEQ ED NO: 10

P2 SEQ ID

Synthetic NO: 11/

SEQ ID NO: 12

KSI-DP-His _t a _g -LL37- DP-His _ta -LL37-ELST-GCN- SEQ ID NO: 5/

ELST-GCN- P1-ELST pET-31b(+)

Pl-ELST -T7 _t SEQ ID NO: 6

KSI-DP-His _tag -LL37- DP-His _ta -LL37-ELST-GCN- SEQ ID NO: II

pET-31b(+)

ELST-GCN- P2-ELST P2-ELST -T7, SEQ ID NO: 8

KSI-DP-His _ta g-ELST- DP-His _ta -ELST-GCN-P2- SEQ ID NO: 19/

pET-31b(+)

GCN-P2-ELST-AEA ELST-AEA-T7 _t SEQ ID NO: 20

Table 2: Legend of genes, function, and number from the database and amino acids/nucleotide sequence, which represents the borders of the used parts of genes.

Swiss Prot Nucleotide Amino acid

Gene name function

No. sequence sequence

T7 _P promoter

T7 _t terminator

HlStag HHHHHH tag

ketosteroid isomerase

KSI AK:1-125

P00947 SEQ ID NO: 21

(SEQ ID NO: 22) protein

dipeptide aspartate- DP GATCCT DP

proline

coiled-coil segment that

PI SEQ ID NO: 9 SEQ ID NO: 10 forms heterodimer with

P2

coiled-coil segment that P2 SEQ ID NO: 11 SEQ ID NO: 12 forms heterodimer with

PI

homodimeric coiled-coil

GCN SEQ ID NO: 13 SEQ ID NO: 14

segment

AK: 134- 170

LL37 P49913 SEQ ID NO: 15 antimicrobial peptide

SEQ ID NO: 16

ELST SEQ ID NO: 17 SEQ ID NO: 18 elastin-like segment

AK: 19-241 (SEQ ID Beta-nerve growth factor NGF P01139 SEQ ID NO: 23

NO: 24) (Beta-NGF) - mouse

silver nanoparticles

AEA SEQ ID NO: 25 SEQ ID NO: 26

forming domain coiled-coil segment that P3 SEQ ID NO: 27 SEQ ID NO: 28 forms heterodimer with

P4

coiled-coil segment that

P4 SEQ ID NO: 29 SEQ ID NO: 30 forms heterodimer with

P3

coiled-coil segment that

P5 SEQ ID NO: 31 SEQ ID NO: 32 forms heterodimer with

P6

coiled-coil segment that

P6 SEQ ID NO: 33 SEQ ID NO: 34 forms heterodimer with

P5

coiled-coil segment that

P7 SEQ ID NO: 35 SEQ ID NO: 36 forms heterodimer with

P8

coiled-coil segment that

P8 SEQ ID NO: 37 SEQ ID NO: 38 forms heterodimer with

P7 Example 2: Production of fusion proteins composed of elastin-like segments with coiled- coil segments, antimicrobial peptide and NGF

Several constructs have been prepared to demonstrate the feasibility of production of fusion proteins listed in Table 1 (SEQ ID No. l, 2, 5, 6).

Plasmids encoding open reading frames of fusion proteins in Table 2 were transformed with chemical transformation into competent E. coli BL21 (DE3) pLysS cells. Selected bacterial colonies grown on LB plates with selected antibiotic (ampicillin) were inoculated into 10 mL of LB broth supplemented with selected antibiotic. After several hours of growth at 37°C 10- 100 iL of the culture were inoculated into 100 mL of selected growth media and left overnight shaking at 37°C. Next day the overnight culture was diluted 20-50-times reaching the OD ₆ oo of diluted culture between 0.1 and 0.2. Culture flasks with 500 ml of diluted culture were put on the shaker and bacteria were grown until OD ₆₀₀ reached 0.6-0.8 when protein expression was induced by addition of inducer IPTG (0.4 mM or 1 mM). Four hours after induction culture broth was centrifuged and bacterial cells were resuspended in lysis buffer (Tris pH 8.0 0.1% deoxycholate supplemented with protease inhibitor coctail) and frozen at - 80°C for at least overnight. Thawed cell suspension was further lysed with sonication and then centrifuged. Precipitate (cell membranes, inclusion bodies) and supernatant were checked for expression of our constructs by SDS-PAGE and Western blot using anti-His-tag antibodies as primary antibodies when necessary. Designed fusion proteins were mainly present in insoluble part (inclusion bodies), which was composed of >80% of the chosen protein. That was due to fusion with KSI domain on N-terminus. Inclusion bodies were washed twice with lysis buffer, twice with 2 M urea in 10 mM Tris pH 8.0 and once with MiliQ water. Usually this treatment resulted in >95% of protein purity. In cases where the precipitated material still contained impurities, inclusion bodies were dissolved in 6 M GdnHCl, pH 8.0, and loaded to Ni -NTA columns. Purification under denaturing conditions was followed according to manufacturer's instructions. After elution with 250 mM imidazole in 100 mM Na ₃ P0 ₄ , 10 mM Tris pH 5.8, fractions containing protein were combined and dialysed twice against 10 mM Hepes pH 7.5 or MilliQ water.

When protein was present in supernatant, supernatant was loaded on Ni ²⁺ -NTA column and purified under native conditions. After elution with 250 mM imidazole pH 5.8 or 500 mM imidazole pH 8.0 fractions containing protein were combined and dialysed twice against 10 mM Hepes pH 7.5 or other appropriate buffer.

Example 3: Preparation of material

Under appropriate conditions proteins with elastin-like segments and coiled-coil segments (parallel heterodimeric or homodimeric) tend to form polypeptide material which can be further employed for eukaryotic cell growth. The coiled-coil segments oligomerize with other coiled-coil segments resulting in enmeshed material.

First screening to find conditions where fusion proteins are soluble were made by dilution of denatured protein in 6M guanidinium-HCl for about 100-times with buffers of various pH (citrate/acetate buffer pH 2 and pH 3, acetate buffers pH 4, pH 5, phosphate buffers pH 5, pH 6, pH 7, Hepes buffer pH 7.5, Tris buffer pH 8, carbonate buffer pH 9, pH 10), different ionic strengths (100 mM, 300 mM, 1 M, 2M NaCl) and different organic solvents up to 20% of acetonitrile, DMSO, methanol or up to 50% in case of trifluoroethanol. Protein absorption spectra were measured and when typical protein spectra were obtained, samples were analysed by CD spectroscopy to determine protein secondary structure and thermal stability when possible. When appropriate conditions were found, denatured protein was dialyzed against selected buffers.

Example 4: Antimicrobial potency of material that embeds antimicrobial peptides

To test the antimicrobial potency of the material that embeds antimicrobial peptides the antibiogram was preformed. The sensitivity of a bacterium E. coli DH5a to antimicrobial peptide was tested with a semi-quantitative way based on diffusion (Kirby-Bauer method).

20 μί, of overnight culture of E. coli DH5a was spread over the 10 cm agar plate (Petri dish) to achieve confluent growth. 6 small discs were then placed on the plate and impregnated with different concentrations of antimicrobial peptide (LL-37) from 0,1-10 mg/ml of protein and MiliQ that served as a negative control. After 16 h of incubation at 37°C the plate was examined for clearing zones. The antimicrobial potency of the material was concentration- dependent (Figure 2.). Example 5: Differentiation of neuronal cells on the polypeptide material composed of two elastin-like segments, two coiled-coil segments and NGF.

The PC 12 rat pheochromocytoma cell line is an established model for nerve growth factor (NGF)-induced neurite formation. It has been shown that when gangliosides are added to the culture medium of PC 12 cells, NGF-induced neurite formation of PC 12 cells is enhanced. Nerve growth factor promotes the survival and differentiation of sensory and sympathetic neurons. It interact with TrkA and low affinity nerve growth factor receptor.

0,25 x 10 ^s PC 12 cells per well were cultivated on μ-slide with 8 wells (Ibidi) after polypeptide material has formed. Growth medium F12K (Gibco) supplemented with 2,5% heat inactivated fetal bovine serum and 15% heat inactivated horse serum was used. Pictures were taken after 48h incubation on 37°C with 5% C0 ₂ atmosphere. Neurite formation is shown on Figure 3D. NGF in the polypeptide material does not affect any morphological changes on HEK293 cells (Human Embryonic Kidney 293 cells) (Figure 3B).

Microscopy of live cells was preformed with Leica TCS SP5 using immersion objective with 63x magnification. Images were further processed with LAS AF 1.8.0. Leica Microsystems.

Example 6: Production of fusion proteins between elastin-like segment and functional polypeptide domain that forms silver nanoparticles with antimicrobial activity

Construct that has been prepared to demonstrate the production of protein with functional polypeptide domain that promote formation of silver nanoparticles is listed in table 1 (No. 7). Production and isolation of protein is the same as in example 2.

Example 7: Antimicrobial potency of polypeptide material that contains functional polypeptide domain that forms silver nanoparticles.

AEA is a designed trimerization coiled coil sequence (SEQ ID: 26) composed of three segments that are combined to form a staggered heterotrimer and include at positions b, c and /of the coiled coil heptad residues Ala, Ala and Glu, respectively. AEA catalyzes formation of silver nanoparticles from silver ions in the solution. AEA fused with elastin-like segment (preparation is described in example 6) is part of the polypeptide material. AEA segment of the material reduces Ag ⁺ to Ag° in form of nanoparticles and after the removal of nonreacted silver ions by dialysis silver nanoparticles remain attached to the matrix. Four different mixtures (table 3) of bacteria (E. coli DC2) were prepared. After overnight incubation (37°C) the OD at 600 nm was measured (Nano Drop) in mixtures no. 1 and 3 as shown on Figure 4.

Table 3 : Mixtures prepared to test antimicrobial potency of polypeptide material that contains functional polypeptide domain that forms silver nanoparticles and Ag-acetate

No. Mixture

1 Dialysed AEA-Ag + LB + E. coli DC2

2 Dialysed AEA-Ag + LB (blank)

3 Dialysed Ag-acetate + LB + E. coli DC2

4 Dialysed Ag-acetate + LB (blank)

Example 8: Tendency of heterodimer formation of coiled-coil segments PI and P2

Coiled-coil segments PI - P8 were designed by inventors. Pairs (P1-P2, P3-P4, P5-P6, P7-P8) tend to form a-helices only in the presence of both components. The principle is illustrated on a P1-P2 pair of coiled-coil segments. Peptides PI and P2 were synthesized by a solid phase synthesis in the Keck center of Yale University, New Heaven, USA. Synthetic coiled-coil segment PI was dissolved in 0.1% ammonium bicarbonate in concentration of 5 mg/ml. P2 was dissolved in distilled water in concentration of 5 mg/ml.

Circular dichroism spectra were recorded on AppliedPhotophysics Chirascan spectropolarimeter (Applied Photophysics, Surrey, UK) under nitrogen flow. Far-UV CD spectra of particular coiled-coil segment as well as coiled-coil segments mixture were recorded between 190 nm and 260 nm in 0.1 -cm path length cuvette using step of 0.5 nm with 1 s/point. Concentration of each coiled-coil segment was 0.1 mg/ml. CD spectrum of individual coiled-coil segment (PI and P2) show the random coil structure. After coiled-coil segments are mixed the spectrum shows a-helix structure (Figure 5).

Example 9: Recovery of the cell from polypeptide material

Upon addition of coiled-coil segment capable of oligomerization with one of the coiled-coil segments of polypeptide material the polypeptide material is easily disassembled. Coiled-coil segment depolymerizes polypeptide material by binding also to coiled-coils segments fused with functional polypeptide domains. Cells are therefore gently released from the matrix without harsh physical conditions, such as temperature, osmotic shock, ionic strength or enzymatic digestion that can affect or damage surface receptors and adhesion molecules.

Polypeptide matrix composed of fusion proteins listed in table 1 (SEQ ID NO: 6, 8) was assembled. After polymerization, the addition of 50 fold molar excess of either PI or P2 resulted in the polypeptide material disassembly.