WO2011046519A1 - Matériau polypeptidique composé de segments de type élastine et segments en super-hélice - Google Patents

Matériau polypeptidique composé de segments de type élastine et segments en super-hélice Download PDF

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WO2011046519A1
WO2011046519A1 PCT/SI2009/000047 SI2009000047W WO2011046519A1 WO 2011046519 A1 WO2011046519 A1 WO 2011046519A1 SI 2009000047 W SI2009000047 W SI 2009000047W WO 2011046519 A1 WO2011046519 A1 WO 2011046519A1
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Prior art keywords
polypeptide
polypeptide material
coiled
seq
elastin
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PCT/SI2009/000047
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English (en)
Inventor
Roman Jerala
Ota Fekonja
Jelka Pohar
Helena Gradisar
Mojca Bencina
Iva Hafner Bratkovic
Robert Bremsak
Spela Miklavic
Urska Jelercic
Anja Lukan
Tibor Doles
Sabina Bozic
Marko Verce
Nika Debeljak
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Kemijski Institut
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Priority to PCT/SI2009/000047 priority Critical patent/WO2011046519A1/fr
Priority to ATA9495/2009A priority patent/AT511187A5/de
Publication of WO2011046519A1 publication Critical patent/WO2011046519A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/73—Fusion polypeptide containing domain for protein-protein interaction containing coiled-coiled motif (leucine zippers)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/735—Fusion polypeptide containing domain for protein-protein interaction containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)

Definitions

  • Polypeptide material composed of elastin-like segments and coiled coil segments
  • 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.
  • 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.
  • biopolymers including proteins such as collagen and fibrin and polysaccharides such as glycosaminoglycans and alginate
  • proteins such as collagen and fibrin and polysaccharides such as glycosaminoglycans and alginate
  • Rationally designed peptides offer a promising way of constructing biomaterials with.
  • 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 ⁇ -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).
  • Extracellular matrix 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.
  • ECM electrospray-like elastic fibres
  • amorphous protein elastin 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 ⁇ -irradiation, chemical cross- linking or the cross-linking can be enzyme-mediated.
  • 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.
  • 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.
  • EGF epidermal growth factors
  • NGF nerve growth factors
  • 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.
  • FIG. 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 5 Circular dichroism spectra of the representative pair of designed coiled-coil segments PI and P2, demonstrating the assembly of heterodimers.
  • 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.
  • polypeptide material therefore mimics extracellular matrix, is biocompatible, directs cellular growth and provides an appropriate environment for cell function.
  • 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) .
  • 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.
  • 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.
  • homologous segments refers also to mutant protein segments, whose mutations minimally alter the amino acid sequence.
  • 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.
  • 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.
  • elastomeric domains having proline and glycine content above threshold are devoid of a-helix and ⁇ -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.
  • 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.
  • 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.
  • coil-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 “ ⁇ 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.
  • 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.
  • 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.
  • 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.
  • the functional polypeptide domain 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.
  • 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.
  • covalently linked has a general meaning and refers to the functional polypeptide domain being bound via peptide bond to the polypeptide material.
  • 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. -
  • 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.
  • 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
  • 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.
  • antimicrobial peptides include, but are not limited to cathelicidins and defensins; preferentially cathelicidin LL-37; preferentially SEQ ID NO: 16.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the invention also encompasses mixing multiple polypeptide materials with different belonging functional parts with the polypeptide materials without functional part.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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-
  • terminal 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.
  • 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.
  • 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.
  • 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.
  • initiation signals for the efficient translation of fusion protein are included, which comprises the ATG and the corresponding sequences.
  • 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.
  • bacterial expression vectors include, but are not limited to: pET vectors, pRSET vectors, and others.
  • the control elements are of bacterial origin.
  • mammalian expression vectors for mammalian cells include, but are not limited to: pcDNA (Invitrogen), pFLAG (Sigma), and others.
  • the control elements are in most cases of viral origin, for example, adenovirus 2, cytomegalovirus, a virus Simian virus 40.
  • the present invention also relates to a host organism expressing the above described proteins.
  • 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.
  • 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.
  • any bacterial host can be used according to the 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.
  • 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.
  • 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.
  • a fusion protein is operatively linked to the signalling sequence that is coded in the nucleic acid.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Protein containing fused functional polypeptide domain with coiled-coil complementary with coiled-coil segments of polypeptide material can be mixed with the polypeptide material.
  • 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.
  • growth has a general meaning and in the description refers to cell/tissue/organ development and cell division.
  • 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.
  • 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 .
  • EGF epidermal growth factors
  • NVF nerve growth factors
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • polypeptide material which also contains functional polypeptide domain, e.g. growth factors
  • functional polypeptide domain e.g. growth factors
  • 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.
  • functional polypeptide domain 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.
  • 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.
  • 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.
  • 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.
  • antimicrobial peptides or metals, particularly silver is their broad-spectrum antimicrobial action.
  • 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.
  • 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.
  • 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.
  • the present invention presents the solution of extenuating or preventing infections, caused by pathogens colonizing prostheses and burn wounds.
  • 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.
  • 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 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 (Imretepark 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.
  • CAMP Homo sapiens cathelicidin antimicrobial peptide
  • 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
  • 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.
  • 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.
  • Precipitate cell membranes, inclusion bodies
  • 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.
  • inclusion bodies were dissolved in 6 M GdnHCl, pH 8.0, and loaded to Ni -NTA columns.
  • proteins with elastin-like segments and coiled-coil segments 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.
  • Example 4 Antimicrobial potency of material that embeds antimicrobial peptides
  • 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.
  • NGF nerve growth factor
  • Example 6 Production of fusion proteins between elastin-like segment and functional polypeptide domain that forms silver nanoparticles with antimicrobial activity
  • 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
  • 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).
  • 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.

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Abstract

L'invention concerne un matériau polypeptidique composé d'au moins un segment de type élastine et d'au moins deux segments en super-hélice et éventuellement d'un domaine polypeptidique fonctionnel et de segments de type élastine représentant au moins quatre répétitions de type élastine. L'invention concerne des applications du matériau polypeptidique pour promouvoir la croissance des cellules, de tissus ou d'organes, la différenciation cellulaire, l'inhibition de la croissance de pathogènes, pour traiter un tissu humain ou animal vivant et pour produire un matériau médical et pharmaceutique qui sera utilisé pour traiter un tissu vivant. DRAWING: Fig. 1 Legend Légende Coiled-coil segment 1 Segment 1 en super-hélice Coiled-coil segment 2 Segment 2 en super-hélice Elastin Segment de type élastine Functional protein domain 1 Domaine de protéine fonctionnelle 1 Functional protein domain 2 Domaine de protéine fonctionnelle 2
PCT/SI2009/000047 2009-10-12 2009-10-12 Matériau polypeptidique composé de segments de type élastine et segments en super-hélice WO2011046519A1 (fr)

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LU501018B1 (en) * 2021-12-15 2023-06-15 Kemijski Institut Nat Institute Of Chemistry Regulation of protein function by insertion of interaction peptides
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