Polypeptide material from elastin-like segments and coiled-coil segments

专利摘要:
The invention provides polypeptide material composed of at least one elastin-like segment and at least two bilayer formation segments, and optionally a functional polypeptide domain. The invention relates to the use of polypeptide material for growth acceleration of cells, tissues and organs, for differentiation of cells, growth inhibition of pathogens, for the treatment of living human or animal tissue, and as medical and pharmaceutical material that will be useful for the treatment of living tissue.
公开号:AT511187A2
申请号:T94952009
申请日:2009-10-12
公开日:2012-09-15
发明作者:
申请人:Kemijski Inst；
IPC主号:

专利说明:

1
¢ 38589) HEL
Polypeptide material composed of elastin-like segments and segments to form double helices
Field of the invention
The field of the invention is novel polypeptide material composed of elastin-like segments, double coil forming segments and, optionally, functional polypeptide domains which define possible uses of the polypeptide material. The field of the invention is a protein that forms polypeptide material and DNK that records a protein that forms polypeptide material.
The field of the invention is also the use of polypeptide material to promote cell growth, tissue or organs, inhibit pathogen growth, and medical / pharmaceutical material useful for healing living tissue, primarily human or animal tissue.
Background Art For the biocompatible basis for cell and tissue growth, various materials have been developed that are important not only for cell culture in vitro, but also for tissue engineering and other tissue engineering applications, as well as for medical applications. Numerous synthetic polymers (for example, polystyrenes, polyethylene-vinyl acetates, polypropylenes, polyethylenes, etc.) and biopolymers (including proteins such as collagen and fibrin, as well as polysaccharides such as glycosaminoglycans, and the like) can serve as the basis of cell division, cell migration, and cell differentiation alginates).
Rationally designed peptides offer a promising approach to the construction of biomaterials. For example, several cases of novel self-assembling and self-complementary amphiphilic peptides with typical exchange of hydrophobic and hydrophilic amino acids have been reported. These peptides form various structures based on the formation of fibrils with β-sheet structures between amphiphiles (US Pat. Appl. Pub. 0209145 A1) and also with biologically active components and / or hydrophobic alkyl tails on terminal parts (US Pat Pub. 0181973 A1, US Pat. 7371719 B2).
Biocompatible materials that mimic extracellular matrix (ECM) represent a favorable environment for cell growth. The extracellular matrix (ECM) is composed of heterogeneous macromolecules, including proteins and polysaccharides that form a three-dimensional environment for cell growth, providing the basis for stabilization and support of cell layers and tissue.
Part of the extracellular matrix are elastic fibers made up of the amorphous protein elastin and microfibrils, mostly composed of fibrillin-1. The precursor of a stable, cross-linked elastin is the soluble molecules tropoelastin. This molecule consists of two distinct domains: hydrophobic domains, rich in amino acid residues glycine (G), valine (V), alanine (A) and proline (P), which often occur in repeat units (the first identified were pentapeptide VPGVG, hexapeptide VGVAPG and Tetrapeptide VPGG) and hydrophilic domains, which are especially rich in alanine and lysine residues, important for cross-linking, which lead to the formation of insoluble and very stable polymer.
Tropoelastin and elastin-related polymers can form co-azervates through hydrophobic domains before molecules covalently cross-link (a process triggered by temperature elevation), which further leads to self-assembly. The tendency for self-aggregation and the important stability of elastin and elastin-like polymers determine elastin crucial component for the development of synthetic nanomaterials.
Lee and coworkers (Lee et al., 2001, Biomacromolecules. 2, 170-179) have thoroughly analyzed polypeptides with elastin-like repeating hydrophobic moieties that can serve as soluble models of elastin. However, cross-linking the polypeptide chains is urgently needed to use these polypeptides as a robust biomaterial. This can be caused by the use of γ-rays, by chemical compounds, or even by enzyme-transferring compounds. Several elastin-like polypeptide materials have already been prepared, where some modifications may be made into hydrophobic repeating sequences, including the replacement of some amino acids with residues that might serve the cross-link, or the addition of components to the crosslink (substrate to lysyl oxidase) of the elastomeric component (Tetra- or pentapeptides, repeating units or their mixture) has introduced (US Pat. 4589882).
In order to achieve a system with better cross-linking, material was also prepared which has simple amino groups at one intermediate of the VPGVG peptide and simple carboxyl groups at the other, allowing cross-linking with a chemical reagent (U.S.Patent No. 4,187,852).
With the use of recombinant technology, multimodular polypeptides based on elastin and capable of modifying mechanical and functional properties of the elastin-based materials have been prepared. The examples include hybrids between elastin-like peptides and the C5 fibronectin domain, which accelerates cell attachment (Welsh et al., 2000, Biomacromolecules. 1,23-30), or hybrids between a peptide-like fibroin of silk and elastin-like peptides (Cappello et al., 1990, Biotechnology Progress. 6, 198-202). Elastin and elastin-like components are used in the field of Na-no biomaterials (which can be a substrate for cell growth or material for delivery of drugs or growth factors) and tissue engineering (also as replacements for skin, blood vessels, heart valves and elastic cartilage) much in view.
Built-up material may also contain various additional functional biologically active portions, which may affect the attachment, adhesion, migration or proliferation of the cells. Attachment of functional biologically active moieties to a scaffold is an important feature in the design of self-assembling peptides. This approach allows us to target the target cell and reduce the amount of substance necessary to achieve the desired local effect. The delivery of some growth factors in soluble form represents an additional disadvantage because the cells no longer respond to the factor because of internalization and because of the regulation in terms of the reduced number of growth factor receptors.
The invention relates to polypeptide material composed of elastin-like segments which are cross-linked to each other via noncovalent interactions using a novel process of selected or designed double helix segments, thus addressing the need for chemical changes to elastin-like repeats avoids introducing cross-connection sites. The invention has been further completed with the use of regulated assembly and disassembly of polypeptide material. The polypeptide material may also be supplemented with added functional protein domains to promote cell growth, differentiation, inhibition of microbial growth, the compound of iron ions, cytotoxicity, cell reprogramming, or many other functions and is therefore novel Biomaterial for the growth of cells / tissue / organs and for the healing of living human or animal tissue dar. Such a method allows almost innumerable possible combinations, also allows attachment of functional protein domains to already built biomaterial after previous construction, which Planning the time course of the therapy allows.
Summary of the invention
The invention relates to polypeptide material which has at least one elastin-like segment and at least two segments for forming double helices. * * * * I * * i * t •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• Contains at least one elastin-like segment between two or more double helix segments, but separate polypeptide molecules combine with each other on interactions between segments to form double helices and are located on these molecules. The invention also relates to polypeptide material optionally containing at least one functional polypeptide domain selected from, but not limited to: growth factors, cell differentiation factors, microbe-repelling peptides, metal-binding domains, and bacterial growth inhibitors. Growth factors were primarily chosen but not limited to: epidermal growth factors (EGF), fibroblast growth factors (FGF) and growth factors that stimulate the growth of neurons, primarily neuronal growth factors (NGF). Microbial-repellent peptides were chosen primarily, but not limited to, cathelicidines and defensins, primarily cathelicidin LL-37. Metal-binding domains are, for example, AEA or hexahistidine tail, preferably SEQ ID NO: 26. One or more functional domains may be covalently bound to polypeptide material, or they may have been inter-segmentated to form double helices connected to the functional domain. and segments for forming double helices that are part of the polypeptide material are introduced into the polypeptide material.
The invention also encompasses polypeptide material composed of various ratios of the polypeptide components, which may also contain different functional domains.
In accordance with the invention, the polypeptide material includes at least one elastin-like segment and at least two double coil forming segments and optionally at least one functional polypeptide domain, wherein the elastin-like segment is human or animal elastin, its mutant or synthetic elastin-like segment having functional elastin properties obtained and which is constituted by at least 4 elastin-like repeats, including but not limited to: pentapeptide Val-Pro-Gly-Val-Gly o -the Gly-Val-Gly-Val- Pro or Gly-Val-Gly-Ile-Pro. In addition, the elastin-like becomes "# *". "".... ""
Segment in this invention characterized in that it is made up of at least 12 amino acid residues, the sum (% (n / n) glycine residues) and 2 * {% (n / n) proline residues) being higher than 60% {n / n).
The invention also relates to the polypeptide material described above, where the number of elastin-like segments is between 1 and 50, preferably between 2 and 10, and the number of segments for forming double helices is between 2 and 50, more preferably between 3 and 10 and where the elastin-like segment for braided double helix formation is interposed at least in one case between two segments.
The invention relates to the polypeptide material described above, where natural or designed motifs for forming double helices represent segments for forming coiled coils and are composed of at least two heptads and are parallel or antiparallel and either homo-oligomers with oligomeric state between 2 and 7 or hetero Form oligomers with oligomeric state between 2 and 7.
The invention relates to polypeptide material where segments for forming double helices connecting different molecules have been selected between designed peptide pairs to form double helices: 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 relates to polypeptide material composed of at least two different polypeptide materials described above, wherein the constructed polypeptide material has been prepared with the compound of at least two polypeptide materials and wherein segments for forming double helices from a material hetero-oligomers having segments to form double helices from a can form other material, whereby we can obtain a regulated compound of assembled polypeptide material. • * · i ··· · I ··· I ···································································· 7
The invention relates to peptides containing double helix segments which are present in only one of the components of the above-described polypeptide material and which we can use to dissect the polypeptide material, allowing for a gentle method of separating the cells from the material.
The invention relates to the polypeptide material described above, wherein segments and domains in the above-mentioned polypeptide material are randomly linked to each other optionally with a linking portion containing one to 20 amino acids, preferably from one to six amino acids, and wherein the protein optionally contains a signal sequence which directs the secretion of the protein and contains (one) amino acid tracer.
The invention relates to the DNA carrying the record for the proteins described above, wherein the DNA is operably linked to regulatory elements, the promoter and the terminator, which allow expression of the fusion protein in the host organism.
The invention relates to the preparation process of the polypeptide material described above, comprising the steps of: a) cultivating a host organism expressing protein which records DNA, both described above; b) the isolation of the expressed protein; and c) the formation of the polypeptide material described above with inclusion of purified protein (s).
The invention relates to the use of the polypeptide material described above for cell or organ growth, which material optionally provides desired cell growth functional properties. The invention also relates to the use of the polypeptide material described above as a medical and pharmaceutical material for the healing of living tissue, primarily of human or animal tissue. For example, but not limited to replacing damaged tissue, such as a prosthesis, for cell regeneration, for reprogramming cells, and as a pharmaceutical material, such as •.
»· · * * Ft * ι 4 I • · # * *» I »· I * · 8 e.g. Cases for the healing of wounds and burns, local delivery of cytotoxic or cytostatic polypeptides.
The invention relates to the use of the polypeptide material described above for growth inhibition of pathogens in the event that the functional polypeptide domains of the polypeptide material are microbial-repellent peptides or metal-binding domains, especially SEQ ID NO: 26, which forms silver nanoparticles.
pictures directory
Figure 1: Schematic representation of the invention. Polypeptide material is composed of elastin-like segments and segments that form double helices. Portions that form double helices oligomerize and link individual polypeptide chains of the polypeptide biomaterial scaffold. Functional polypeptide domains may be integrated into the polypeptide chain as part of the fusion protein, or linked to the scaffold via interactions between segments that form bilobed scaffolds and those attached to the functional polypeptide.
Figure 2: Microbe repellent effect of polypeptide material containing a functional polypeptide domain - microbe-rejecting peptide LL-37. Shown in the picture are the following patterns: 1-MilliQ, 2-6 increasing concentrations of polypeptide material containing a functional polypeptide domain - microbe-rejecting 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: Differentiation of PC12 cells on polypeptide material containing a functional polypeptide domain - growth factor NGF. The picture shows: [A, B] HEK293T cells on polypeptide material [A] without functional polypeptide domain, [B] with a functional polypeptide domain - the growth factor NGF; [C, D] PC12 cells on polypeptide material [C] without functional polypeptide domain, [D] with functional polypeptide domain - the growth factor NGF.
Figure 4: Microbe-repellent effect of polypeptide material containing the functional polypeptide domain AEA, which forms silver nanoparticles. The picture shows that a functional polypeptide domain that forms silver nanoparticles inhibits bacterial growth.
Figure 5: The CD spectrum proves the formation of heterodimers of a representative pair of planned segments that form a double helix, P1 and P2.
Detailed disclosure of the invention
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly accepted by one of ordinary skill in the art. The terminology used in the description of the invention is intended to be a description of a particular part of the invention and not of limitation of the invention. All publications mentioned in the description are listed under references.
In the description of the invention and in the claims, the single number is described, but the description also includes a plurality, which is not emphasized for ease of understanding.
polypeptide material
The invention is based on the discovery that at least one elastin-like segment having at least two segments to form double helices can form polypeptide material. The illustrated invention describes polypeptide material whose construction in the field of nanomaterials is based on recent events by inventors. The invention is based on the discovery that protein composed of at least one elastin-like segment and at least two double coil forming segments forms polypeptide material having specific physical properties, e.g. Promote growth of cells, tissues or organs. Elastin-like segments mimic elastin, provide elastomeric properties, and are linked by the oligomerization of the segments to form double helices, resulting in the formation of polypeptide material. Polypeptide material therefore mimics the extracellular matrix, is biocompatible, directs cell growth, and provides a proper environment for cell function. • * * · 4 · * · 10
In the specification, the term "polypeptide material" refers to the material constructed of proteins containing at least one elastin-like segment and at least two segments for forming double helices and whose structure is two- or three-dimensional. Segments resembling elastin material provide elastomeric properties, while double helix segments enable inter-connection of elastin-like segments.
The inventors have recognized that elastin-like repeats can be linked to polypeptide material with a novel method of using the segments for the formation of double helices that oli-merge. This is an improvement on the previous inventions because previously it was necessary to modify the crosslinking of elastin-like repeats with the introduction of possible cross-linking sites (U.S. Patent No. 4,598,882, U.S. Patent No. 4,187,852).
The term "elastin-like segment" has a general meaning in the description of the invention and refers to homologous portions of animal and human elastin composed of at least 4 elastin-like repeats. Elastin-like segments may be strung together or, at least in one case, may be attached to form double helices between two segments. The term "elastin-like segment" also refers to mutated or synthetic, engineered, elastin-like segment that retains the characteristics of elastin. Elastin-like segments can be expressed chemically synthesized or recombined. The term "homologous portions" as used in the specification refers to the amino acid sequence of the protein derived from the same or different organism and closely resembling in protein balance, preferably greater than 50% of the resulting structure, primarily 60%, more preferably 70% % in the balance of the sequence. The term "homologous sections" also refers to mutated protein sections whose mutations minimally alter the sequence of amino acids.
The term "elastin-like repeats" also refers to hydrophobic repeating sequences in elastin that contain a lot of Gly (G), Val (V), Ala (A), * * * »« · · tt · * ·· * ♦ and Pro (P) residues often occur in tandem repeats of a few (from 3 to 6) amino acids. The term "elastin-like repeats" includes, but is not limited to, pentapeptides having the sequence 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 made up of at least 12 amino acid residues, the sum (% (n / n) glycine residues) and 2 * (% (n / n) proline residues) being higher than 60% (n / n). The elastomeric function of elastin depends on only two amino acid residues - glycine and proline. These properties of elastin make themselves felt above a certain limit buzzer of proline and glycine. The second property of elastomeric domains with a glycine and proline content above the mentioned limit is the absence of the α-helix and the β-sheet, the polypeptide remains in its unregulated form, even 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, preferably between 2 and 10, and the number of segments for the formation of double helices between 2 and 50, preferably between 3 and 10, wherein the elastin-like segment is incorporated in at least one case between the segments to form double helices. However, the double helix segments form homo-oligomers or hetero-oligomers with an oligomerization state between 2 and 7.
The term "homo-oligomerization" given in the description of the invention has a general meaning and refers to the formation process of the complexes constructed from a single monomer type, the number of monomers being between 2 and 7.
The term "hetero-oligomerization" given in the description of the invention has a general meaning and refers to the formation process of the
Complexes composed of different types of monomers, with the number of monomers between 2 and 7.
As used in the above description, the term "monomer" refers to a segment that can form the double helix and integrate with another segment that forms the helix by winding both.
The term " segment to form double helices " has a general meaning and refers to structural protein motifs composed of 2 or more α-coils, which wrap around and thus form a supercooling coil. These motifs contain heptad repeats, labeled (a-b-c-d-e-f-g) n, each two helical turns. &Quot; a " and " d " usually do not represent polar, hydrophobic amino acid residues located on the interface between the coils, " e " and " g " are polar amino acid residues that are exposed to the solvent and interact electrostatically, " b ", " c " and " f " but are hydrophilic and exposed to the solvent. Different amino acid residues at the sites " a-g " determine the oligomerization state, the specificity, the orientation of the helix and the stability. More specifically, the term " double helix formation segment " as used in the specification refers to natural or planned structural motifs of the protein which form coiled coils and contain at least two heptads and can be parallel or anti-parallel and can form homo- or hetero-oligomers.
Segments for forming double coils may be selected from natural or designed structural motifs of the proteins to form double helices, primarily from natural proteins with leucine closure, e.g. BZIP or designed sequences for the formation of double helices, primarily 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 relates to the discovery that the polypeptide material, in addition to the structural role of important domains for the formation of double helices and
Elastin-like domains additionally containing at least one functional polypeptide domain, which gives the polypeptide material additional functional properties, such as the promotion of cell growth and its differentiation, the growth inhibition of pathogens or the binding of metal ions.
A functional polypeptide domain may be covalently bound to at least one elastin-like segment having at least two segments to form double helices forming polypeptide material.
In addition to the covalent attachment of the functional domain to the polypeptide material as described above, the invention also relates to a second ability to integrate the polypeptide domain into the polypeptide material. The inventors have discovered that it is useful to be able to introduce functional properties into polypeptide material that does not contain a functional domain. The inventors have discovered that polypeptide material can be added to a functional polypeptide domain that is combined with the double helix segment, and thus the functional polypeptide domain, combined with the double helix segment, is formed by the oligomerization of complementary segments to form double helices. Wendeln involved in the polypeptide material in the polypeptide material.
In this way, we can add additional desired properties to the polypeptide material already containing a functional polypeptide domain with the addition of another polypeptide functional domain associated with the double helix segment that is complementary to the segment for forming double helices in the polypeptide material. Functional polypeptide domains may be added to polypeptide material at the beginning of construction or post-addition, and in addition we may introduce them into the organism at various sites, but still aiming for the site of the implanted polypeptide material having complementary segments to form double coils.
When the functional polypeptide domain is covalently linked to the polypeptide material, it is accessible to the cells, tissue or organ. The local concentration of a functional polypeptide domain is higher because of the absence of diffusion than in the case of a soluble polypeptide functional domain. In addition, to achieve the desired local effect, a lower functional polypeptide domain is necessary than in the case of a domain in soluble form. In addition, potential toxicity and adverse effects in this manner are limited to the area of the implanted polypeptide material. Such a type of material also allows a temporal change in therapy with sequential introduction of various functional proteins.
The term " united (merged) or fusion " has a general meaning, but the description refers to the process of preparing the hybrid protein from two previously separated proteins / domains / segments, optionally linked to a link containing from one to 20 amino acids.
The term " covalent bonding " has a general meaning and refers to functional polypeptide domains that are linked to the polypeptide material with a peptide bond.
The term " complementary " refers to the ability of one segment to form double helices that homo- or hetero-oligomerizes with another segment to form double helices.
The term " functional polypeptide domain " refers to molecules that introduce any functional properties into the polypeptide material, such as promotion of cell growth, proliferation inhibition of bacteria, differentiation of cells, or binding of metal ions. The polypeptide functional domain may include but is not limited to: growth factors, microbe repellent peptides, and metal ion binding domains.
The term " growth factor " has a general meaning and refers to the molecule that binds to cell receptors and controls the growth, proliferation or differentiation of the target cells or tissue. Examples of 15
Growth factors include, but are not limited to: epidermal growth factor (EGF), fibroblast growth factor (FGF), neuronal growth factor (NGF), erythropoietin and others; primarily NGF, primarily SEQ ID NO: 24.
The term " microbe-repellent peptide " refers to a broad-spectrum antibiotic that is bactericidal but also acts to cause damage to the membrane, metabolism, or targeting of cytoplasmic components to fungi or viruses. Examples of microbe-rejecting peptides include, but are not limited to: Kathelizidine and De-fensine; primarily the Kathelizidin LL-37, with priority SEQ ID NO: 16.
The phrase " metal binding domains " has a general meaning and refers to peptides that can bind metals, including, but not limited to, Ag, Au, Pd, Pt, which has a microbe-repelling effect. The domain for binding metals is primarily SEQ ID NO: 26, which is composed of 3 sections capable of trimerization and forms silver nanoparticles.
The invention also includes the recognition that polypeptide material also containing a functional polypeptide domain is covalently bound to a polypeptide material, which may also be a mixture (i) of polypeptide material containing at least one elastin-like segment and at least two segments Formation of coiled coils; and (ii) polypeptide material which also contains a functional polypeptide domain in addition to at least one elastin-like segment and at least two double helix segments.
With regard to the desired properties of the polypeptide material, the mixture may be composed of various ratios, with polypeptide material containing not only at least one elastin-like segment and at least two double helix segments but also a functional polypeptide domain, less than 50% of the mixture, more specifically 20%. and most often 1-5% · * »· t« «t» ·
•. Polypeptide materials may be mixed due to molarity, mass, volume, etc.
Such heterogeneous mixtures may have certain advantages over homogeneous mixtures. Namely, homogenous mixtures contain only polypeptide material which contains at least one elastin-like segment and at least two segments for forming double helices, which does not ensure desired properties, such as the promotion of cell growth, cell adhesion, growth inhibition of bacteria, cell differentiation, metal-ion bonds, etc. On the other hand, however, homogeneous mixtures containing only polypeptide material containing, in addition to at least one elastin-like segment and at least two double helix segments, also a functional polypeptide domain, form material which is at least one of the polypeptide material Elastin-like segment and at least two segments designed to form double helices is weaker.
In order to combine the desired properties of polypeptide material composed of various related functional polypeptide domains, the invention also encompasses the mixing of multiple polypeptide materials with different related functional domains with polypeptide materials that do not contain polypeptide domains.
Polypeptide material which can be disassembled using peptide containing a segment to form double coils
The basis of the invention is also the discovery that polypeptide material can be dissected with the addition of peptide containing a segment to form double helices and is also present in the polypeptide material.
The inventors have discovered that the peptide supplement, which contains a segment for forming double helices and resembles the segment for forming double helices in polypeptide material, is a method by which one obtains P o... decomposes lypeptidmaterial. The added double helix segment is integrated into the structure of the polypeptide material because of the ability to form oligomers with one of the segments to form double helices in polypeptide material, thereby loosening polypeptide material resulting in its decomposition.
In this way we can separate cells growing on polypeptide material of the mentioned material without resorting to chemical changes (eg pH, ionic strength), physical changes (eg long incubations at high temperatures, osmotic shock) or enzymatic degradation of polypeptide material Affect or damage surface receptors and adhesion molecules, or induce stress in cells. So obtained cells can be counted, transplanted or used for further biochemical analyzes or technological applications.
The term " peptide containing a segment for forming double helices " in the specification refers to a segment for forming double helices, wherein the segment having one of the segments for forming double helices in the polypeptide material is capable of oligomerization. The term " segment to form double helices " refers to segments for forming double helices described above.
Polypeptide material composed of at least two different polypeptide materials
The basis of the invention is also the discovery of the polypeptide material, which is a combination of at least two different polypeptide materials. Composite polypeptide material is prepared by combining two polypeptide materials, wherein segments for forming double helices from one of the materials can form hetero-oligomers having segments to form double helices in the other material. The inventors have discovered that the hetero-oligomerization of segments to form double helices is made of a material with Seg ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• In order to form co-coils of a different material, it is a way of controlling the onset of the composition of the polypeptide material. Namely, soluble polypeptide material that contains a segment for forming double helices and is capable of hetero-oligomerization does not assemble when it is the only polypeptide material in the solution. The formation of polypeptide material may possibly be triggered only with the addition of another material containing the double helix segment and hetero-oligomerized with the segment to form bilayers of the first material, which provides a mechanism for directing the material.
Composite material contains at least two polypeptide materials, but it may also be composed of several, even up to ten, more often from two to eight different polypeptide materials relating to the invention.
Protein that assembles polypeptide material
The description refers to the protein of the polypeptide material described above. The protein contains at least one elastin-like segment and at least two double helix segments, with at least one elastin-like segment sandwiched between two or more bilateral helix segments, and individual polypeptide molecules sharing interactions among bilayer helix segments are bound to these molecules. In addition, the protein comprising polypeptide material may also contain a functional polypeptide domain, which may also be part of the protein. When the functional polypeptide domain is part of the protein that assembles polypeptide material, it is covalently bound and can not diffuse away, which is an important advantage.
The invention relates to protein containing unified double helix segments and a functional polypeptide domain. The inventors have discovered that the functional polypeptide domain can be integrated into the polypeptide material via interactions of the segments to form double helices with complementary segments to form bilayers present in the material. "" Φ · · Φ I * | f · « * * * «* Φ I * * * * * * * Φ ·« · · Φ φ · φ Φ «19. In this way, functional properties can be added to the material.
Segments and domains of the protein described above may optionally be separated from one another by one to twenty amino acids, preferably one to six amino acids. Proteins may optionally contain a signal sequence that directs the secretion of the protein and amino acid marker for purification and detection of the protein.
The expression " link " refers to shorter amino acid sequences, whose task is only the separation of individual protein domains or segments. The role of the linker, which may be included in the protein of choice, may also include the introduction of injection sites for post-translational modifications, including the introduction of sites for better processing. The length of the link is not limited, although it is usually up to 30 amino acids long, preferably one to 20 amino acids, most often one to six amino acids. Any amino acids may be included in the linker, but primarily amino acids are chosen among, but are not limited to: serine, glycine, threonine, proline, valine, and alanine residues, which are only available through double helix or elastin segments -like domains allow flexibility and specific intermolecular association.
The expression " signal sequence " used in the specification. refers to the sequence of amino acids necessary to direct the protein to a specific site in the cell. The signal sequence depends on the host organism in which the fusion protein is expressed. The amino acid sequence of signal sequences is well known to experts, as well as which signal sequence is functional in a particular organism.
The term " amino acid marker " refers to sequences of amino acids added to the protein intentionally to facilitate purification, isolation or detection of the protein. • * * 4 I »* * · ····> 4 * I ·
»« »· I * I» AI 20
The location of the signal sequence, linkers, and amino acid labels is optional, but it must allow for the functional expression of the protein and maintain the function for which these amino acid sequences have been selected. DNA that records protein from polypeptide material
The invention also relates to the DNA which records protein of polypeptide material containing at least one elastin-like segment and at least two segments to form double coils, with at least one elastin-like segment between two or more segments to form double helixes and wherein individual polypeptide molecules join to one another via interactions between segments to form double helices on these molecules and are optionally linked to a functional protein domain, but segments of the polypeptide are optionally linked together by a linker. The protein optionally contains a signal sequence and amino acid markers. The specification also refers to DNA which records protein from polypeptide material containing unified segments to form double helices and polypeptide functional domains, segments and domains optionally linked together with a linker, but the protein optionally also contains signal sequences and amino acid tags.
The term " DNA " refers to the nucleotide sequence in the open reading frame which records the protein which is the subject of the invention and is operatively linked to regulatory elements, a promoter and terminator, allowing expression of the protein in host cells. The length of the DNA sequence can be very different, depending on the individual protein.
The term " regulatory elements " used in the specification. has a general meaning and refers to the DNA region in the expression vector where regulatory proteins bind, e.g. Transcription factors, and control gene expression. »
The term " promoter " has a general meaning and refers to the DNA region in the expression vector that allows for a copy of the target gene. The promoter type depends on the host organism in which the gene is expressed. In the case of a pronunciation in prokaryotes, e.g. E. coli, the use of several promoters is possible, e.g. Plac, PT5, priority PT7 is used. In case of a statement in eukaryotes, e.g. in mammalian cells, promoters are most often derived from viruses, e.g. PCMV or PSV40.
The term " terminator " has a general meaning and refers to the sequence that marks the end of the gene to be copied. The terminator type depends on the host organism in which the gene is expressed. In the case of a pronunciation in prokaryotes, e.g. E.coli, the terminator from bacteriophage T7 can be used.
Recombinant nucleic acid
The invention is based on standard molecular biology methods well known to those skilled in the art (see Sambrook et al., 1989. Molecular Cloning: A laboratory manual, 2nd Ed., Cold Spring Harbor, NY, Ausubel et al. tocols in Molecular Biology, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., NY).
The described polypeptide material proteins can be synthesized by expressing the DNA that labels these proteins in a suitable host organism. DNA that records proteins is inserted into a corresponding expression vector. Appropriate vectors include, but are not limited to: plasmids, viral vectors, etc. Expression vectors compatible with host organism cells are well known to those skilled in the art and include appropriate controls for transcription and translation of the nucleotide sequence. Typically, an expression vector will include an expression cassette linked in 5 'to 3' direction from a promoter, a fusion protein coding sequence operatively linked to regulatory elements, a promoter and a terminator, including the RNA polymerase stop codon and a polyadenylation signal for the polyadenylation is composed.
The expression vector can be prepared for expression in prokaryotic or eukaryotic cells. An example of prokaryotic cells are bacteria, e.g. Escherichia coli. According to the invention, prokaryotic cells are used to obtain a sufficient amount of nucleic acid. The expression vector usually contains control elements operably linked to the DNA of the invention carrying the record for proteins. Controls are chosen to produce an effective and tissue-specific expression. The promoter may be constitutive or inducible, depending on the desired utterance pattern. The promoter may be derived from a host organism or else of foreign origin (located in cells where we use it), it may be natural or synthetic. The promoter must be selected so that it acts in target cells of the host organism. Also included are signals for the onset and efficient translation of the fusion protein - ATG and associated sequences. When the vector used in this invention includes two or more reading frames, they must be independently connected to the control elements. However, the controls may 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 bacterial cells, the control elements are of bacterial origin.
Examples of mammalian mammalian cell expression vectors include, but are not limited to: pcDNA (Invitrogen), pFLAG (Sigma) and others. When vectors are used in mammalian cells, in most cases the control elements are derived from viruses, e.g. the adenovirus 2, the cytomegalovirus, the virus Simian Virus 40. 23
host organism
The invention also relates to the host organism which expresses the proteins described above.
The term " host organism " refers to the organism in which the DNA that protein registers to express itself has been used. The introduction of vectors into host organisms is carried out by conventional methods known to those skilled in the art. These methods include: transformation, transfection, including: chemical transformation, electroporation, microinjection, DNA lipofection, cell sonication, microbombing, DNA virus introduction, and others. According to the invention, the DNA with transformation is introduced into bacterial cells.
The host organism can be pro- or eukaryotic. Eukaryotic cells capable of expressing the fusion protein are not limited in use as long as the cell lines are compatible with the methods of propagation of the expression vector and with the expression of the fusion protein. Preferred eukaryotic cells include, but are not limited to: yeasts, fungi, plant cells and mammalian cells such as: mouse, rat, monkey or human fibroblast cells.
According to the invention, any bacterial host can be used to express the DNA. To express proteins of this invention, the use of bacteria or yeasts is best suited. The protein may express itself in bacteria E. coli or B. subtilis or in yeasts S. cerevisiae or P. pastoris. The preference bacterium is E. coli. The invention relates to bacteria or yeasts that express protein, primarily to bacteria E. coli or yeast fungi P. pastoris.
Production of the Protein / Process for Preparing Polypeptide Material The invention also relates to the process of producing polypeptide material and includes introducing the DNA which confers the protein of the polypeptide material into the host organism, the cultivation of the host organism. Suitable conditions for expression of the protein, isolation of the protein and exposure of the protein to specific conditions with the intention of forming polypeptide material are recorded.
Fusion protein can be synthesized in the host organism expressing heterologous nucleic acid that detects fusion protein. Fusion protein of this invention is used to prepare polypeptide material. The fusion protein may be linked by a signal sequence encoded in the nucleic acid.
In general, the heterologous nucleic acid is included in the expression vector (virus or no virus), as described above.
The invention encompasses host cells or organisms containing nucleic acid (transient or stable) which carries the record for the fusion proteins described above. Suitable host organisms are known to experts in the field of molecular biology and include bacterial and eukaryotic cells.
The introduction of vectors into host cells is carried out as described above in conventional, known methods.
The introduction of DNA can be transient or stable. A transient statement refers to the introduction of the DNA vector that is not introduced into the genome of the cell. A stable infiltration is achieved by the introduction of DNA into the host genome. DNA transfer, especially in the preparation of the host organism with a stable DNA integration, can be monitored with the presence of markers. DNA that records markers refers to the resistance in the fabric, e.g. Antibiotics, and may be included in the vector containing genes for fusion proteins, or in a separate vector.
The protein is expressed in a corresponding host organism. Expression of large amounts of fusion protein can be found in bacteria such as E. coli, and "f". "" I "*." "" "* *" "F *." "* Reach for yeast fungi P. Pastoris; In addition, cultures of mammalian cells can be used for the production of lower levels of protein, where post-translational modifications are important for proper stacking and function.
It is known that protein can be expressed in cells of the following organisms: humans, rodents, cattle, pigs, poultry, rabbits, and the like. Host cells can be cultivated as primary or immortal lines. The expression of the fusion proteins may be constitutive or inducible, e.g. the expression of the protein can be activated by the addition of the inducer IPTG, which triggers the expression of the desired protein because of the binding of the lac repressor. The fusion protein can be expressed in soluble fraction or in inclusion bodies.
Techniques for purifying the protein include chromotography with size separation, ion (exchange) chromotography, reverse phase chromatography and affinity chromatography when proteins or attached labels react specifically with a purification column. These techniques are known to experts of this area.
Compositions of polypeptide material are achieved under favorable conditions that allow bilin-helical segments to connect elastin-like segments. When polypeptide material is composed of a single type of protein, we find suitable conditions by examining the solubility and secondary structure of the soluble protein part, and more particularly the precipitate, in different pH buffer (usually pH 3 to pH 9), ionic strength, organic solvents (eg DMSO, acetonytril, trifluoro-ethanol). Important factors in self-assembly are the concentration of the fusion protein and the temperature. Polypeptide material is formed when the concentration of the protein is between 0.1 mg / mL and 20 mg / mL, usually from 0.5 to 10 mg / mL. Self-assembly of polypeptide material may also be activated by heating the precipitate of the fusion protein above the lowest melting point temperature followed by slow cooling. ««
When polypeptide material is composed of several different fusion proteins, self-assembly normally begins when proteins are mixed in a suitable molar ratio in conditions favorable for self-assembly.
The protein of the polypeptide material containing at least one elastin-like segment and at least two double helix segments may be mixed in different proportions with the protein which also contains a functional polypeptide domain. With regard to the desired properties of the polypeptide material, the mixture may be prepared from a variety of ratios, with the protein containing, in addition to at least one elastin-like segment and at least two double coil forming segments, also a functional polypeptide domain, less than 50% of the mixture, more specifically % and most often represents 1-5% of the mixture. By mixing two proteins, wherein segments to form double helices of one protein form hetero-oligomers with segments to form double helices of another protein, the onset of polypeptide material formation can be controlled.
Protein containing a functional polypeptide domain combined with the double helix segment complementary to the segment to form double helices in polypeptide material may be added to the polypeptide material as well.
By incorporating the functional moieties that are combined with the helix forming segments, functional properties can be added to the polypeptide material. With the addition of peptides composed of double helix segments present in one of two materials comprising polypeptide material, degradation of polypeptide material that allows easy recovery of cells from the material can be achieved. 27
Application of the polypeptide material
The invention also relates to possible uses of the polypeptide material. The basis is to be able to use polypeptide material as a material / pharmaceutical preparation for cell, tissue or organ growth, cell differentiation, cell repair, cell reprogramming, cytotoxic function, for medical and pharmaceutical material for living human or animal tissues, for the growth prevention of pathogens.
The term " growth " has a general meaning and refers to the development of cells / tissues / organs and cell division. The illustrated invention provides an environment for cultivating any cell type, including, but not limited to, smooth muscle cells, fibroblasts, keratinocytes, epithelial, immune, and endothelial cells. Polypeptide material replicates the natural environment of cells - extracellular matrix, because it includes a network of elastin-like segments. Cells, tissues and organs can be cultured on the surface of the polypeptide material, in addition, the cells can spread in its structure or, in the case where the material forms a three-dimensional structure, migrate into the material itself.
Cells, tissues and organs can be cultured on polypeptide material that is similar to the physiological conditions as they can be cultured in a suitable cell culture container. Depending on the cell proliferation, their number and density, which depends on the cell type and the purpose of use, cells can be cultured on Polypeptidmaterial correspondingly long.
In the case of cell culture on polypeptide material which additionally contains at least one or more combinations, usually 2 to 10, functional polypeptide domains, include, but are not limited to, growth factors, cell adhesion molecules, molecule chemotaxis, ligand receptors, microcells, and the like. robotic peptides, factors for reprogramming, factors for cell differentiation, cytotoxic or cytostatic polypeptides, such as epidermal growth factor (EGF), fibroblast growth factors, neuronal growth factors (NGF), especially SEQ ID NO: 24, microbe-rejecting peptides such as cortelizidines and defensins; primarily the Kathelizidin LL-37, with priority SEQ ID NO: 16.
It is therefore not necessary to add functional polypeptide domains since this is already bound to the polypeptide material on which cells grow, which is a great advantage. As already mentioned, there is also the possibility of mixing polypeptide material with different functional polypeptide domains in different ratios, which makes it possible to guarantee the desired properties for optimum cell growth and offers almost innumerable possible combinations.
The addition of peptides constituting a segment for forming double helices incorporated in polypeptide material may result in the degradation of the material, which presents the possibility of a gentle removal of cells which are then counted, further transplanted or in further biochemical analysis or various technological applications. In contrast to the known methods, which include biochemical and physical changes or the decomposition of enzymes. The mentioned method does not affect or damage molecules on the cell surface or cause stress to the cells.
Polypeptide material can be used as a material for medical and pharmaceutical purposes, for healing of living tissue, especially human and animal tissue.
Polypeptide material, with or without cells or tissue growing on surfaces, can be used as a material to treat numerous injuries or tissue diseases, and can be transplanted into the body through surgical or other appropriate procedures.
The term " treatment " has a general meaning and in the specification refers to the method of troubleshooting damaged or injured human or animal tissue using polypeptide material. ··· * * * * * * * * * * * * * * * * t * · i ··· · · »• #« ································································· ♦ ♦ "* 29
The term " preparation of medical and pharmaceutical material " refers to methods which we use to control polypeptide material in such a form or to prepare it in such a condition as to provide e.g. as a replacement for injured tissue, as a prosthesis or as a pharmaceutical material, e.g. Cases for the treatment of wounds and burns, can be applied.
The described polypeptide material causes minimal or barely perceptible immune resp. Inflammatory reactions. In general, the polypeptide material described in the invention can be used in any situation involving injury or damaged tissue resulting from surgery, trauma, tumor, degenerative or other disease. Polypeptide material can be used to repair natural elastic systems, especially those containing elastin and tropoelastin, by replacing the damaged part of the system, e.g. Ligaments, tendons, blood vessel walls, and the like, with polypeptide material. Polypeptide material can also be surgically transplanted to a site of damaged or missing vascular material in humans or animals, and is thus applicable as vascular replacement or closure of lesions. Polypeptide material is useful for reconstituting the structural and / or functional integrity of the tissue.
Cells that have proliferated / differentiated on the polypeptide material can be removed from the structure and further cultured in containers for cell cultures in vitro. Later, it will be able to relay it to the individual and apply it as a substitute tissue or organ.
The use of polypeptide material which also contains a functional polypeptide domain, e.g. Growth factors for the treatment of living human or animal tissue have certain advantages over treatments using growth factors in soluble form. Soluble growth factors may have undesirable effects, especially in vivo, e.g. Stimulating the growth of cells that target target cells, causing these cells to pass through target cells, or diffusion into the bloodstream and expression of new effects elsewhere. With the addition of a functional polypeptide domain to the polypeptide material, we achieve a local effect of the functional domain on target cells. The described material offers a potential way of inhibiting the growth of undesired tissue with local cytotoxic or cytostatic action at the site of application, especially but not limited to cancerous tissue and the like.
When epithelial cells are placed with the intention of reconstructing an epidermis part, polypeptide material may serve as a skin basis for transplantation to the recipient. Polypeptide material is also applicable to the construction of prostheses containing human-like elastin, e.g. Tubes as a replacement for blood vessels and sheaths for the treatment of wounds and burns. A prosthesis can become a permanent tissue replacement because patient cells, including endothelial cells, invade the polypeptide material. Polypeptide material may also be used to cover surfaces of any type of prosthesis, including prostheses made of synthetic materials and / or metals.
The term " prosthesis " refers to any material implanted in the body, including the replacement of blood vessels, heart valves, or patches of material. It can also be applied as a sheath that stimulates the treatment of wounds and burns.
The rugs have also discovered that polypeptide material, if it contains, in addition to at least one elastin-like segment and at least two double helix-forming segments, also a functional polypeptide domain which is a microbeneploying peptide, such as cathelizidines and defensins; in particular, the ka-thelizidine LL-37, especially SEQ ID NO: 16, or a metal-binding domain, especially SEG ID NO: 26, which forms silver nanoparticles, is also applicable to the growth inhibition of pathogens. The advantage of microbe-repellent peptides and metals, especially silver, is in their broad spectrum of microbe-repelling function. 31
The term " pathogens " refers to common bacteria, fungi, yeasts and viruses that cause disease. In particular, it refers to common bacterial pathogens such as, but not limited to, one gram of positive bacteria S. aureus, S. pyogenes, S. pneumonia, and one gram of negative bacteria H. influenzae, K. pneumoniae, L. pneumophila , P. aeruginosa, E. coli.
Infections often occur as a result of any intervention in the tissue. In view of the fact that Polypeptidmateria! As a substitute for damaged or damaged natural elastic tissue and applied to the recipient, it is a particular advantage if polypeptide material also inhibits the growth of pathogens and thus alleviates or prevents potential infection. In addition, polypeptide material is also applicable to the construction of prostheses, e.g. of tubes as a replacement for blood vessels or sheaths for other applications such as the treatment of wounds and burns, as already mentioned, or for covering prosthetic surfaces. Prostheses often present a danger to the patient due to the development of biofilms, which can lead to many infections that are dangerous to humans, including infections caused by e.g. P. aeruginosa and S. aureus, two bacteria that are often found in biofilms and also burns infections. The invention described in this context provides a way to alleviate and prevent infections caused by prostheses and burns present pathogens.
In view of the fact that silver is not selectively and extremely abundantly active in small quantities, the rasters have discovered that polypeptide material that contains a domain for metal binding, especially SEG ID NO: 26, forms silver nanoparticles and is a particularly promising material for the Growth inhibition of pathogens, particularly applicable for bandaging wounds and for prostheses. • · «« «· ·« * · * «··· I« I · * «« · · · · · · · · 32
To illustrate the invention, examples of methods are given below. The descriptions of the examples are not intended to limit the invention and should be construed as a demonstration.
Examples of the methods
Example 1: Preparation of the DNA Construct
The polypeptide material described herein has been engineered to combine elastin-like segments, double helix segments, and functional domains. Amino Acid Sequences of Segments to Form Heterodimeric Parallel Double Coils (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 constructed on the basis of facts about the composition of double helix segments known to those skilled in the art, with the intention of increasing the specificities of the interactions between the double helix segments. The amino acid sequence of the segments to form homodimeric parallel coiled coils was taken from the peptide GCN4 (SEQ ID: 14). The trimerization sequence to form the double helix AEA (SEQ ID: 26), which forms silver nanoparticles, was constructed from facts about the composition of double helix segments known to those skilled in the art, with repetition of three segments which combine into a step-shaped heterotrimer and have, at the sites of heptades b, c and f, double coils of amino acid residues Ala (b), Ala (c) and Glu (f).
Elastin-like segments were selected from repeats of the pentapeptide (SEQ ID: 18).
The functional domains selected for this example are: the neuronal growth factor (SEQ ID: 24), the microbe-rejecting peptide LL-37 (SEQ ID: 16). They were selected from the natural protein sequences of rodents and humans.
33
Fusion proteins may include a signal sequence to localize the protein and a peptide label to detect or purify the protein. DNA sequences optimized for expression of the protein in the desired host (E. coli) were generated by amino acid loading and Gene Designer DNA 2.0 1.0 Version 1.0.0.1 (DNA2.0 Headquarters 1430 O'Brien Drive, Suite E, Menlo Park, CA 94025, USA). DNA sequences were ordered from MR.Gene GmbH (Gewerbepark B32, D-93059 Regensburg) and excised with residual endonucleases. The LL-37 DNA sequence was taken from the open reading frame plasmid of the clone of the species Homo sapiens cathelicidin microbe repellent peptide (CAMP). Part of the gene encoding LL-37 was multiplied by PCR. DNA constructs and associated fusion proteins are described in Table 1. Detailed descriptions and functions of individual DNA and / or protein products are described in Table 2. All DNA constructs have a start codon (ATG) before the histidine sign. The constructs were cloned with a high level of expression of the peptide sequence connected to the protein of the ketosteroid isomerase in the vector pET-31b (+). The expression cassette contains in the direction 5 'to 3' the promoter T7, the encrypted sequence KSI and a multiple cloning site for the fusion protein and the terminator T7. These regulatory elements allow the expression of the protein in prokaryotic cell line E. coli, which expresses the T7 RNA polymerase. DNA constructs were prepared using molecular biology techniques described in the Molecular Biology Handbook (Sambrook J., Fritsch EF, Maniatis T. 1989. Molecular cioning: A laboratory manual, 2nd Ed., New York, Cold Spring Harbor Laboratory Press: 1659 p.). Plasmids, constructs and intermediates were chemically transformed into bacteria E. coli DH5a or BL21 (DE3) pLysS. 34
Fusion proteins associated with the engineered DNA constructs are generally composed of at least one elastin-like segment and at least two segments to form double coils, and optionally also contain a functional polypeptide domain (e.g., NGF and / or LL-37). The KSI-DP domain allows expression of proteins in inclusion bodies and acid cleavage in aspartate-proline (DP) compounds.
Table 1: Fusion proteins used to demonstrate the invention
nucleotide /
No. Name Construction of the construct Amino acid sequence Plasmid 1 KSI-DP-Histag-LL37 ELST-GCN-ELST DP-Hista-LL37-ELST-GCN-ELST-T7t SEQ ID NO: 1 / SEQ ID NO: 2 pET-31b (+) 2 KSI-DP-Histag-LL37-ELST-GCN-P2-ELST-NGF DP-Hista-LL37-ELST-GCN-P2-ELST-NGF-Th SEQ ID NO: 3 / SEQ ID NO: 4 pET -31b (+) 3 PI synthetic SEQ ID NO: 9 / SEQ ID NO: 10 4 P2 synthetic SEQ ID NO: 11 / SEQ ID NO: 12 5 KSl-DP-His (ag-LL37-ELST-GCN-Pl) ELST DP-Hista-LL37-ELST-GCN-Pl-ELST-T7, SEQ ID NO: 5 / SEQ ID NO: 6 pET-31b (+) 6 KSI-DP-HiStag-LL37-ELST-GCN-P2-ELST DP-His, α-LL37-ELST-GCN-P2-ELST-T7, SEQ ID NO: 7 / SEQ ID NO: 8 pET-31b (+) 7 KSI-DP-His, ag-ELST-GCN-P2- ELST-AEA DP-Hisla-ELST-GCN-P2-ELST-AEA-T7t SEQ ID NO: 19 / SEQ ID NO: 20 pET-31b (+)
Table 2: List of genes, functions and numbers from the database as well as amino acid / nucleotide sequence, which is a limit of the applied genetic parts. 35
Name of the Gene Swiss Prot No. Nucleotide sequence Amino Acid Sequence Function T7p Promoter T7t Terminator Histag HHHHHH Amino Acid Marker KSI P00947 SEQ ID NO: 21 AK: 1-125 (SEQ ID NO: 22) Ketosteroid Isomerase DP - GATCCT DP Dipeptide Aspartate Proline Segment to form a PI " SEQ ID NO: 9 SEQ ID NO: 10 A double helix forming heterodimer with P2 Segment to form a double helix forming heterodimer with PI. GCN SEQ ID NO: 13 SEQ ID NO: 14 Double helix segment LL37 P49913 SEQ ID NO: 15 AK: 134-170 SEQ ID NO: 16 Microbe-rejecting peptide ELST SEQ ID NO: 17 SEQ ID NO: 18 Elastin-like segment NGF PO1139 SEQ ID NO: 23 AK: 19-241 (SEQ ID NO: 24) Beta neuron growth factor (beta-NGE) - from mice AEA - SEQ ID NO: 25 SEQ ID NO: 26 Domain for formation of silver nanoparticles segment for formation P3 - SEQ ID NO: 27 SEQ ID NO: 28 A double helix forming heterodimer with P4 Segment for formation of a P4 - SEQ ID NO: 29 SEQ ID NO: 30 A double helix which forms heterodimer with P3 Segment for formation P5 - SEQ ID NO: 31 SEQ ID NO: 32 A double helix forming heterodimer with P6 Segment for forming a P6 - SEQ ID NO: 33 SEQ ID NO: 34 ner double P7 - SEQ ID NO: 35 SEQ ID NO: 36 A double helix forming heterodimer with P8 segment to form a P8 " P8 " SEQ ID NO: 37 SEQ ID NO: 38 a double helix which forms heterodimer with P7
Example 2: Preparation of the fusion protein composed of elastin-like segment and segments to form double coils, the microbe repellent peptide and NGF
Several constructs were prepared to prove the possibility of producing fusion proteins listed in Table 1 (SEQ ID NO: 1, 2, 5, 6).
Plasmids with recordings (in open reading frame) for fusion proteins listed in Table 2 were chemically transformed into the competent E. coli BL21 (DE3) of the pLysS cell. Selected bacterial colonies grown on LB plates with selected antibiotic (ampicillin) were inoculated into 10 ml of liquid LB cultures with added selected antibiotic. After several hours of growth at 37 ° C, we inoculated 10-100 L of culture in 100 mL of selected growth medium, which was then shaken overnight at 37 ° C. The next day, we diluted the culture 20 to 50 times, resulting in an OD600 of the diluted culture between 0.1 and 0.2. Culture containers with 500 ml of the diluted culture were attached to the shaker, the bacteria were cultured until OD600 reached 0.6 0.8. The expression of the protein has been induced with addition of the inducer IPTG (0.4 mM or 1 mM).
Four hours after induction, we centrifuged the broth, resuspended bacterial cells in the lysine buffer (Tris pH 8.0, 0.1% deoxycholate with added mixture of protease inhibitors) and frozen at -80 ° C at least overnight. The thawed cell suspension was further lysed with sonication followed by centrifugation. The deposit (cell membrane, inclusion body) and supernatant were checked by SDS-PAGE and Western blot analysis using anti-His-tag antibodies as primary antibodies, if necessary, to confirm the expression of the constructs. The engineered fusion proteins were mostly present in the insoluble part {inclusion body), which consisted of > 80% of the desired protein were composed. This was the result of the fusion with the KSI domain at the N-terminal part of the protein. The inclusion bodies were washed twice with lysine buffer, twice with 2M Ureo in 10 mM Tris pH 8.0 and once with MiliQ water. The result was usually > 95% pure protein. If the deposit still contained impurities, we dissolved the inclusion bodies in 6 M GdnHCl, pH 8.0, and applied to a Ni 2+ NTA column. Drying under denaturing conditions proceeded according to the manufacturer's instructions. After elution with 250 mM imidazole in 100 mM Na 3 PO 4, 10 mM Tris pH 5.8, we combined the fractions containing protein and dialyzed twice against 10 mM Hepes pH 7.5 or MilliQ water.
If the protein was found in the supernatant, we applied the supernatant to the Ni2 + -NTA column and purified it under native conditions. After elution with 250 mM imidasol pH 5.8 or 500 mM Imidasol pH 8.0, we pooled the fractions containing protein and dialysed twice against 10 mM Hepes pH 7.5 or other appropriate buffer.
Example 3: Preparation of the material
Under appropriate conditions, the protein with elastin-like segments and double helix segments (parallel heterodimers or homodimers) tends to form polypeptide material that can be used to grow eukaryotic cells. Segments to form double helices oligomerize with others, resulting in the formation of cross-linked material.
In searching for conditions in which fusion proteins are soluble, we diluted the denatured protein in 6M Guanudinium HCl approximately 100 times, with buffers of different pH (citrate-acetate buffer pH 2 and pH 3, acetate buffer pH 4, pH 5, phosphate buffer pH 5, pH 6, pH 7, buffer Hepes pH 7.5, buffer Tirs pH 8, carbonate buffer pH 9, pH 10), different ionic strength (100 mM, 300 mM, 1 Μ, 2M NaCl) and in various organic solvents with up to 20% acetonytril, DMSO, methanol or up to 50% trifluoroethanol. We measured the absorbing spectrum of the protein. In the case of typical protein spectra, we have analyzed the pattern with CD spectroscopy to establish secondary structures of the protein and temperature stability. When appropriate conditions were determined, we dialyzed the denatured proteins against selected buffer.
Example 4: Microbe repellent function of the material containing microbe-repellent peptides »38
The microbe-repellent function of material containing microbe-repellent peptides has been verified with the antibiogram. The susceptibility of the bacterium E. coli DH5a to microbial-repellent peptide was tested by the semi-quantitative method based on diffusion (Kirby-Bauer method). 20 L of the overnight culture of E. coli DH5a was spread over a 10 cm diameter agar-agar plate (Petri dish) to achieve confluent growth. We then placed 6 smaller slices on the plate and impregnated them with various concentrations of microbial-rejecting peptide (LL-37) material of 0.1-10 mg / ml protein and MiliQ, which served as a negative control. After 16 hours incubation at 37 ° C, the plate was checked for clarification zones. The microbe-repellent function depended on the concentration of the material (Fig. 2).
Example 5: Differentiation of nerve cells on polypeptide material composed of two elastin-like segments, two segments to form double coils and NGF
The cell line of the rat Feochromocytoma PC12 is an established model for the formation of neurites induced by neuronal growth factor (NGF). It has been demonstrated that when gangliosides are added to the medium of the PC12 cells, this accelerates the formation of the neurites induced by the neuronal growth factor (NGF). The neuronal growth factor helps in the survival and differentiation of soreness and sympathy neurons. It affects TrkA and the receptor of low affinity neuronal growth factor receptor (LNGFR).
The experiment was carried out in microscopy chambers with 8 holes each (Ibide). After formation of polypeptide material, we inoculated PC12 cells at a concentration of 0.25 x 10 5 cells per well. We have added the growth medium F12K (Gibco) supplemented with 2.5% temperature-inactivated fetal bovine serum and 15% temperature-inactivated horse serum. The images were taken 48 hours after incubation at 37 ° C with 5% CO 2 in the atmosphere.
The formation of neurites is shown on image 3D. NGF in polypeptide material does not cause morphological changes to HEK293 cells (human embryonic kidney 293 cells) (Figure 3B).
We examined living cells under the Confocal Microscope Leica TCS SPS on Leica DMI 60000 CS Stands. We used a 63x oil immersion lens. We have the pictures with the program LAS AF 1.8.0. Leica Microsystem won.
Example 6: Production of fusion protein between an elastin-like segment and a functional polypeptide domain that forms silver nanoparticles with microbial repellency of activity.
The construct prepared for the purpose of producing the functional polypeptide domain protein, which causes silver nanoparticles to form, is shown in Table 1 (entry 7). The production and isolation of the protein is the same as in Example 2.
Example 7: Microbe repellent effect of polypeptide material containing a functional polypeptide domain for formation of silver nanoparticles. AEA is a constructed trimerization sequence to form a double helix (SEQ ID: 26). It consists of three segments, which combine into a step-shaped heterotrimer and have at the sites of heptad b, c and f double helices of amino acid residues Ala (b), Ala (c) and Glu (f).
The AEA catalyzes the formation of silver nanoparticles from silver ions in solvents. AEA combined with an elastin-like segment (the preparation is described in Example 6) is part of the polypeptide material. The AEA segment of the material reduces Ag + in AgO in the form of nanoparticles. After removal of unreduced silver ions by dialysis, silver nanoparticles remain attached to the matrix.
40
We prepared four different mixtures with bacteria (E. coli DC2) (Table 3). At incubation (37 ° C) overnight, we measured OD at 600 mm (nano drop). The value of OD600 blends 1 and 3 is shown in Figure 4.
Table 3: Mixtures prepared for verification of microbial-rejecting function of polypeptide material containing a functional polypeptide domain for formation of silver nanoparticles and silver acetate
No. Mixture 1 diaiysed AEA-Ag + LB + E. coli DC2 2 diaiysed AEA-Ag + LB (without bacteria) 3 dialyzed Ag-acetate + LB + E. coli DC2 4 dialyzed Ag-acetate + LB (without bacteria)
Example 8: Tendency to Form Heterodimeric Segments to Form Doppler Coils P1 and P2
Segments to form double helices P1 to P8 have formed the indicia. Pairs (P1-P2, P3-P4, P5-P6, P7-P8) form the α-coiled coil only if both components are present. The principle is shown on pair P1-P2. The two peptides P1 and P2 were synthesized by synthesis with a stable phase, at KeckZetrum, Yale University, New Heaven, USA. The synthetic segment for formation of double helix P1 was dissolved in 0.1% ammonium bicarbonate to a final concentration of 5 mg / ml. The P2 was dissolved in distilled water to a final concentration of 5 mg / ml.
The CD spectrum was recorded with a spectro-polarimeter under nitrogen flow (Applied Photophysics, Surrey, UK). CD spectra of the broader UV region of the respective segments to form double helices and their mixtures were measured in the cell by optical means 0.1 cm every 0.5 nm in the range of wavelengths 190nm - 260 nm, wherein the time of measurement at a wavelength of 1 Second fraud. The concentration of the segments for the formation of * * * * * * * * * * * * * * * * * * * * * * · * · · · · · · · · * * * * 4 «·» * «* · ♦ · 41
Double helices were 0.1 mg / ml. The CD spectrum of the respective double helix segments {P1 and P2) shows a random protein structure. In the case where the two segments were mixed to form the double helix, the spotting tower has an a-helical structure.
Example 9: Recovery of cells from polypeptide material
With the addition of the segment to form double coils and being capable of oligomerizing with the formation of a coiled coil with one of the segments of the polypeptide material, polypeptide material readily degrades. The added segment also depolyses polypeptide material to bind to double helix segments and connect them to functional polypeptide domains. The cells are so gently released from the matrix without the use of harsh physical conditions such as temperature, osmotic shock, ionic strength or enzyme degradation that affect or damage surface receptors and adhesion molecules.
The polypeptide matrix was composed of fusion proteins listed in Table 1 (SEQ ID NOs: 6, 8). After polymerization, the result of adding 50-fold molar overproduction P1 and P2 was degradation of the polypepid material. 42
FOLLOW <210 &gt; 1 &lt; Al &gt; 960
&Lt; 212 &gt; DNA &lt; 213 &gt; Synthetically &lt; 220 &gt; &Lt; 221 &gt; CDS &lt; 222 &gt; (1) . , (960) &lt; 223 &gt; KSI-DP-Histag-LL3 7-EL3T-GCN-ELST &lt; 400 &gt; 1 acg cat acc cca gaa cac atc acc gcc gtg gla cag cgc ttt gtg gct Met 1 H is Thr Pro Glu 5 His Ile Thr Ala Val 10 Val Gin Arg Phe Val 15 Ala gcg ctc aat gcc ggc gat ctg gac ggc atc gtc gcg ctg ttt gcc gat Ala Leu Asn Ala 20 Gly Asp Leu Asp Gly 25 I le Val Ala Leu Phe 30 Ala Asp gac. gcc acg gtg ga a cac gtg ggt tcc gag ccc agg tcc ggt acg Asp A1 a Th r 35 Val Glu Asp Pro Val 40 Gly Ser Glu Pro Arg 45 Ser Gly Th r gct gcg att est gag ttt tac gcc aac teg ctc aaa ctg cct ttg gcg Ala Ala 50 Ile Arg Glu Phe Tvr 55 Ala Asn Ser Leu Lys 60 Leu Pro Leu Ala gtq gag ctg acg cag gag gta cgc gcg gtc gcc aac gaa gcg gcc t LC Val 6 5 Glu Leu Thr Gin Glu 70 Val Arg Ala Val Ala 75 Asn Glu Ala Ala Phe 80 gct ttc. acc gtc agc tr.c gag act cag ggc cgc aag acc gta gtt gcg Ala Phe Thr Val Ser 85 Phe Glu Tyr Gin Gly 90 Arg Lys Thr Val Val 95 Al a ccc atc gat cac ttt cgc ttc aat ggc gcc ggc aag gtg gtq agc atc Pro Ile Asp Hi s 100 Phe Arg Phe Asn Gly 105 Ala Gly Lys Val Val 110 Ser Ile cgc gcc ttg trt ggc gag aag aat att cac gca tgc cag atg gat cct Arg Ala Leu 115 Phe Gly Glu Lys Asn 120 Ile His Al a Cys G1 n 125 Met Asp Pro atg cat cac cat cac cat cac et aga gcc ggc ctg ctg ggt gat Met Tyr 130 His H is His His K is 135 Hi s Ser Arg Ala Gly 140 Leu Leu Gly Asp ttc ttc egg aaa ta aaa gag aag att ggc aaa gag ttt aaa aga old Phe 145 Phe Arg T.vs Ser Lys 15 0 Glu lys Tie Gly Lys 155 Glu Phe Lys Arg τ le 1 60 gtc cag aga atc aag gat tr.t Gtg egg aat ctc gta ccc agg aca gag Val Gin Argile Lys 165 Asp Phe Leu Arg Asn 170 Leu Val. Pro Arg Thr 175 Glu tcc tcc ggg gtL ccg ggt gtt ggt gtt cct ggc gtg ggt gtt cct ggt Ser Ser Gly Val 180 Pro Gly Val Gly Va 1 185 Pro Gl y Val Gly Val 190 Per Gly gtt ggc gcg gtg gga gt g cc.a ggc gtt ggt. Val Gly Val 195 Pro Gly Val Gly Val 200 Pro Gly Val Gly Val 205 Pro Gly Val gggcgggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggg V7 a 1 Gly Va 1 Pro Gly Val Gly 48 96 144 192 240 288 336 384 432 480 5 28 576 624 672 ····· II t · * »• i * ·· t - * m * * • I ·« « »# * · 210 215» t 43 * «* * 22C m * a * gtt ccg optional gcc tggg gt ag aaa cag ctg gaa gat aaa atc 720 Val 225 Pro Gly Val Gly Ser 230 Glv Arg Ket Lys Gin 235 Leu Glu Asp Lys Ile 240 gag gag ctg ctg tct aag atc tac cac ctg gaa aac gaa att get ege 768 Glu Glu Leu Leu 5er 245 Lys Ile Tyr His Leu 250 Glu Asn Glu Ile Ala 255 Arg ctg aaa aag ctg att ggt gaa ege LCC according to gtt gcgg gtt ggt 816 Leu Lys Lys Leu 260 Ile Gly Glu Arg Ser 265 Gly Val Pro Gly Val 270 Gly Val cct gggggg gtt cct ggt gtt ggc gtg cca ggt gtg gga gtg cca 864 Pro Gly Val 275 Gly Val Val Gly Val 280 Gly Val Pro Gly Val 285 Gly Val Pro ggc gLL greggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggg agt 960 Val Gly 305 &lt; 21C &gt; &lt; 211 &gt; &lt; 21 2 &gt; &lt; 21 3 &gt; &Lt; 400 &gt; Val Pro Gly 2 320 PRT Synthetic 2 Val 310 Gly Val Pro Gly Val 315 Gly Ser Gly Thr Ser 320 MeL 1 Hls Thr Pro Glu 5 His ile Thr Al a Val 10 Val Gin Arg Phe Val 15 Ala A_ Leu Asn Ala 20 Gly Asp Leu Asp Gly 25 Ίθ Val Al a Leu Phe 30 Ala Asp Asp Ala Thr 3 5 Val Gl u Sp Sp Pro Val 40 Gly Ser Gl u Pro Arg 4 5 3er Gly Thr Ala Ala 50 Ile Arg Glu Phe Tyr 55 Ala Asn Ser Leu Lys 60 Leu Pro Leu Ala Val 65 Glu Leu Thr Gin Glu 70 Val Arg Ala va 1 Ala 75 Asn Glu Ala Ala Phe 80 Ala Phe Thr Val Ser 85 Phe Glu Tyr Gin Gly 9C Arg Lys Your Val Va 1 95 Ala Pro I le Asp H is 100 Phe Arg Phe Asn Gly 105 Ala Gly Lys va * Val 110 Ser Ile Arg Ala Leu 115 Phe Gly G1 u Lys Asn 120 Ile His Ala Cys Gin 125 MeL Asp Pro .Met Tyr] 30 His His His H is His 135 His Ser Arg Ala Glv 140 Leu Leu Gly Asp Phe 145 Fhe Arq lys Ser Lys 150 Glu Lys Ile Gly Lys 1 55 Glu Phe Lys Arg Ile 1 60 Va &quot;. Gin Arg 1 Le Lys 165 Asp Phe Leu Arq Asp. 170 Leu Val Pro Arg Thr 175 Glu 44
Gly Val Gly Val 180 Pro Gly Val Gly Val 190 Pro Gly Val Gly Val 190 Pro Gly Val Gly Val 195 Pro Gly Val Gly Val 200 Pro Gly Val Gly Val 205 Pro Gly Val Gly Val 210 Pro Gly Val Gly Val 215 Pro Gly Val Gly Val 220 Pro Gly Val Gly Val * 225 Pro Gly Val Gly Ser 230 Gly Arg MeL Lys Gin 235 Leu Glu Asp Lys Ile 240 Glu Glu Leu Leu Ser 245 T, vs I le Tyr His Leu 250 Glu Asn Glu Ile Ala 255 Arg Gly Val Pro Gly Val 285 Gly Val Pro Gly Val 290 Gly Val Pro Gly Val 295 Gly. Leu Ly s Lys Leu 260 Ile Gl y Glu Arg Ser 265 Gly Val Pro Gly Val 270 Gly Val Pro Gly Val 275 Val Pro Gly Val 300 Gl.y Val Pro Gly Val 3C5 Gly Val Pro Gly Val 310 Gly Val Pro Gly Val 315 Gly Ser Gly Thr Ser 320 &lt; 210 &gt; &Lt; 211 &gt; &Lt; 212. &gt; &lt; 213 &gt; - &gt; 1740 DNA Synthetic ≤ 220 &gt; &lt; 221 &gt; &Lt; 222 &gt; &Lt; 223 &gt; CDS {D. , (1740) KS: -DP-Histag-LL37-ELST-GCN-P2-ELST-mNGF &lt; 400 &gt; atq eat MeL His 1 3 acc Thr cca Pro gaa Glu 5 cac His alc ile acc Thr gcc Ala gtg Va 1 10 g La Val cag Gin ege Arg ett Phe gtg Val 15 gct Ala 48 gcg Ara ctc Leu tiat Asn gcc Ala 20 ggc Glygate Asp ctg Leu gac Asp ggc Gly 25 atc 71e gtc Va] gcg Ala ctg Leu ett Phe 30 gcc Ala gat Asp 96 gar. Asp gcc A1 a arg Thr 35 gtg Val gaa Glu gac Asp ccc Pro gtg Val 4C g Gly clcc Glu ccc Pro agg Arg 45 tcc Sergt Gly aeg Thr 1 44 gct Ala gcg A 1 a 50 att Tween Arg gag Glu tr. T Phe cac Tyr 55 gcc Ala ädC Asn teg Ser ctc Leu ddä Lys 60 ctg Leu cct Pro ttg Leu gcg Al a 192 gtg Val 65 gag Glu ctg Leu aeg Thr cag Gin gag Glu 70 gta Va_ege Arg gcg Ala gtc Val gcc Ala 75 aac Asn gaa Glu gcg Ala gcc Ala ttc Phe 80 240 gct Ala tr.c Phe acc Thrgtc Val agc Ser 8 5 t tc Phe gag Glu tat Tyr cag Gin ggc Gly 90 ege Arg Cl CVL Lys acc Thr gta Val gtt Val 95 gcg A 1 a 288 c.cc Pro acc Ile gat Asp cac His ICO did Phege Arg ctc Phe aat Asn ggc Gly 105 gcc Al a ggc Gly aag Lys gtg Val gtg Val 1 1 0 agc Ser atc I le 336 45 cgc gcc ttg ttt ggc gag aag aat et t cac gca tgc cag at.g gat cct 384 Arg Ala Leu 115 Phe Gly Glu Lys Asn 120 Ile Hls Al a Cvs Gin 125 Met Asp Pro ctg did cat cac cat cac cat cac tet aga gcc ggc ctg ctg ggt gat 432 Met. Tyr 130 H is His His Hls H is 135 His Ser Arg Ala Gly 140 Leu Leu Gly Asp LtC tt.c egg aaa ta aaa gag aag att ggc aaa gag Üt 3 ci a aga aLt 480 Phe 145 Phe Arg T.ys Ser Lys 150 Glu Lys Ile Gly Lys 155 Glu Phe Lys Arg Ile 160 gtc cag aga atc aag gat ttt ttg egg aaL ett q La ccc agg aca gag 528 Val Gin Arg Ile Lys 165 Asp Phe Leu .Arg Asn 170 Leu Val Pro Arg Thr 175 Glu tcc Lee ggg gtt. ccg ggt gtt ggt gtt cd. gggt ggt gtt cd. 576 Ser Ser Gly Val 180 Pro G1 y Val Gly Val 185 Pro Gly Val Gly Val 190 Pro Glygtt qgc g Lg cca ggt qtg gga gtg cca ggc gtt ggt gta ccg qgt gtg 624 Val Gly Val 1 95 Pro Gly Val Gly Val 200 per Gly Val G j. y Val 205 Pro Gly Val gggcgggggggggggggggggggggggggggggggggggggg. ggt ggg 672 Gly Val 210 Pro G '. y Val Gly Val 21 5 Pro Gly Val Gly Val 220 Pro Gly Val Gl y gtt ccg qgt gta ggt tcc ggg egt atg aaa cag ctg gaa gat aaa atc 720 val 225 Pro Gly Val Gly Ser 230 Gly Arg Met Lys Gl π 235 Leu Glu Asp Lys Ile 240 gag gag ctg ctg tot aag atc cac cac ctg gaa aac gaa att get 768 Glu Glu Leu Leu Ser 2 45 Lys lie Tyr Hi s Leu 250 Glu Asn Glu T le Ala 255 Arg ctg aaa aag ctg att gta cqc tcc ggg LCt cca gsa gac aaa atc 816 Leu Lys Lys Leu 260 Ile Gly Glu Arq Ser 265 Gly Ser Pro Glu Asp 270 Lys Ica gca cag ctg aaa gaa a ag aac gcc ctg 3 3 c'i Qa3 aag aat 03 3 cag 864 Ala Gin Leu 275 Lys Glu I ys Asn Ala 280 Ala Leu Lvs Gl u Lys 285 Asn Gin Gin ctg aag gag β 3 3 atc ca a gca ctg aaa did ggc t &quot; o c ggg gtt ccg ggt 912 Leu Lys 290 Glu Lys i Gin A-Lcj 295 Leu Lys Tyr Gly Ser 300 G1 y Val Pro Gly gt.L gg ί gtt cct gggg gtt cct ggt qtt ggc gtg cca qgt. gtg 960 Val 3C5 a ly Val Pro Gly Val 310 Gly Val Pro Gly Val 315 Gl v Val Pro Gly Val 3 20 gga gt-g c ca ggc gtt ggt gta ccg ggt gtg ggc gtt ccg gga gtc gga 1008 Gly Val Pro Gly Va 'Gly Val Pro Gly Va L 330 Gly Va' Pro Gly val 335 Gly qtt ccg gga gta ggc gtg cct. ggt gtt ggg gtt ccg ggt gta ggt tcc 1056 Val Pro Gly Va 1 340 Gly Vai Pro Gl y Val 345 Gly Val Pro Gly Val 350 Gly Ser ggg qaa ccg act act gat aqc aac gtg cca gaa ggc gat agc gta cca 1104 Gly Eq u Pro 355 Tyr Thr. Asp Ser Asn 360 Val Pro G lu Gly Asp 365 Ser Val Pro Q itlggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggg 1 1 52 Glu Ala 3 7C Hi s rrp Th r:, ys Leu 375 G n H.i s Ser Leu Asp 3 8C Thr AI a Leu Ara 46 «* ·· 4 &lt; * ··· ···························································································································································································································································································· Val Thr Gly 400 c.ag acr. egt aat atc aeg gtg gct cct egt ctg ttc aaa aaa ege ege 1248 Gin Thr Arg Asn Ile 405 Thr Val Asp Pro Arg 410 Leu Phe Lys Lys Arg 415 Arg ctg cat tet cct egt gtt ctg ttt agc aca caa cct ccg cca aca tca 1296 Leu His Ser Pro 420 Arg Val Leu Phe Ser 425 Thr Gin Pro Pro 430 Thr Ser agc gat aca ctg gac ctg gac ttt caa gca ca L gga acc att ccg ttc 1344 Ser ASp Thr 435 Leu Asp Leu ASp Phe 440 Gin Ala His Gly Your 445 Ile Pro Phe aat egt acc cat egt tca aaa ege tca agc acc cat ccg gtg ttt cat 13 92 Asn Arg 450 Thr His Arg Ser Lys 455 Arg Ser Ser Thr His 460 Pro Val Phe His arg ggg gaa ttt teg gtt tgt gac agc gtg Let gtc tgg gtt ggg gat 1440 Met 465 Gly Glu Phe Ser Val 470 Cys Asp Ser Val Ser 475 Val Trp Va &quot;. Gly Asp 480 aaa acc aca g acc alc aaa ggc aaa CJ α cl gtg acc gtc ctg gcc 1488 Ly s Thr Thr Ala Thr 485 Asp Ile Lys Gly Lys 490 Glu Val Thr Val Leu 495 Ala gaa gtc aat acc aac aac agc gtc ttt egt caa tat ttc ttc gaa acc 1536 Glu Val Asn 500 e 500 Asn Asn Ser Val Phe 505 Arg Gin Tyr Phe Phe 510 Glu Thr aaa tgc egt gcc tca aat cct gta gaa agc qqg tgt egt gga att gat 1584 lys Cy S Arg 515 Ala Ser Asn Pro Val 520 Glu Ser Gly Cys Arg 5 25 Gly Ile Asp Lea aaa cat tgg aac teg act acc acc act cac acc r tc gtt aaa 1632 Ser Lys 53 0 His Trp Asn Ser Tyr 53 5 Cys Thr Thr Thr His 540 Thr Phe Val Lys gcc ctg act acc gac gag aaa caa get get tgg ege ttt atc egt atc 1 680 Ala 545 Leu Thr Thr Asp Gl u 550 Lys g: n Ala Ala Trp 555 Arg Phe Tie Arg Ile 56C gat acc get tgt gtg egt gtc ctg tcc cg L aaa gca aca egt egt ggt 1728 Asp Thr tcc gga Ser G'_ y &lt; 210 &gt; &lt; 211 &gt; &lt; 21 2 &gt; &Lt; 213 &gt; &Lt; 400 &gt; Ala Cys Val 565 act agL Thr Ser 580 4 580 FRT Synthetic 4 Cys Val Leu Ser Arg 57 0 Lys Ala Arg Arg 575 Gly 1740 Met 1 His Thr Pro Glu 5 His I le Thr Ala Val 10 Val Gin Arg Phe Val 15 Ala ALa Leu Asn Ala 20 Gly Asp Leu Asp Gly 25 Ile Val Ala Leu Phe 30 Ala Asp * * «* *« * 47 * · Λ «* * • · 4 4 Asp Ala Thr 35 Val Glu Asp Pro Val 40 Gly Ser Glu Pro Arg 45 Ser Gly Thr Ala Ala 50 Tie Arg Gl u Phe Tyr 55 A .1 a Asn Ser Leu Lys 60 Leu Pro Leu Ala Val 65 Glu Leu Thr Gin Glu 70 Val Arg AJ a Val Ala 75 Asn Glu Ala Ala Phe 80 Al a Phe Thr V a1 Ser 85 Phe GLu Tyr Gin Gly 90 Arg Lys Thr Val Val 95 Al a Pro Ile Asp His 100 Phe Arg Phe Asn Gly 105 Ala Gly Lys Val Val 110 Ser Ile Arg Al a Leu 115 Phe Gly Glu Iy s Asn 120 ile His A1 a Cvs Gin 125 Met Asp Pro Met Tyr 130 His Hi His His His 135 His Ser Arg Ala Gly 140 Leu Leu Gly Asp Phe 145 Phe Arg Lys Ser Lys 150 Glu Lys Ile Gly Lys 15 5 Glu Phe Lys Arg Ile 160 Val Gin Arg Tie Lys 165 Asp Phe Leu Arg Asn 170 L Eu val Pro Arg Thr 175 Glu Ser Ser Gly Val 180 Pro Gly Val Gly V a 1 185 Pro Gly Val Gly Val 1 90 Pro Gly Val Gly Val 1 95 Pro Gly Val Gly Val 200 Pro Gly Va 1 Gl y Va 1 205 Gly Val Gly Val 210 Pro Gly V ac Gly Val 215 Pro Gly Val Gl y Val 220 Pro Gly Val Gly Val 225 Pro Gly Val Gly Ser 230 Gly Arg Met Lys Gin 23 5 Leu Glu Asp Lys Ile 240 Glu Glu Leu Leu Ser 245 i &gt; y 3 Tie Tyr His Leu 25 0 Glu Asn Glu I le Ala 25 5 Arg Leu Lys Lys Leu 260 Ile Gly G1 u Arg Ser 265 Gly Ser Pro Glu Asp 270 Lys I Ala Gln Leu 275 Lys Glu Lys Asn Al a 280 Ala Leu Lys Glu Lys 285 Asn Gin Gin Leu Tys 290 Gl u Lys TI e Gin Ala 295 Leu Lys Tyr Gly Ser 3 00 Gly Val Pro Gly Va 1 305 Gly val Pro Gly val 310 Gly Val Pro Gly val Gly Val Pro Gly Val 320 Gl y Val Pro Gly Val 3 25 Gly Val Pro Gly Val 330 Gly Val Pro Gly Val 335 Gly Val Pro Gly Val 340 Gly Val Pro Gly Val 34 5 Gly Val Pro Gly Val 350 Gl y Ser GJ y Gl U Pro 355 Tyr Thr Asp Ser Asn 360 Val Pro Glu Gl y Asp 365 Ser Val Pro Glu Ala 370 His p rp Thr Lys Leu 375 Gln Hi s Ser Leu Asp 380 Thr Ala Leu Arg
Arg Ala Arg Ser Ala Pro Thr Ala Pro Ile Ala Ala Arg Val Thr C-ly "* 48 385 390 395 400 Gin Thr Arg Asn I le 405 Thr Val Asp Pro Arg 41 0 Leu Phe Lys Lys Arg 415 Arg Leu His Ser Pro 420 Arg Val Leu Phe Ser 425 Thr Gin Pro Pro 430 Thr Ser Ser Asp Thr 435 Leu Asp Leu Asp Phe 440 Gl n A1 a His Gly Thr 445 Ile Pro Phe Asn Arg 450 Thr His Arg Ser Lys 455 Arg Ser Ser Thr Hi s 460 Pro Val Phe His Net 465 Gly Glu Phe Ser Val 470 Cys Asp Ser Val Ser 475 Val Trp Val Gly Asp 480 Lys Thr Thr Ala Thr 485 Asp Ile Lys Gly Lys 490 Gl u Val Thr Val Leu 495 Ala Glu Val Asn I le 500 Asn Asn Ser Val Phe 505 Arg Gin Tyr Phe Phe 510 Glu Thr lys Cys Arg 515 Ala Ser Asn Pro Val 520 Gl u Ser Gly Cys Arg 525 Gly Ile Asp Ser Lys 530 His Trp Asn Ser Ivr 535 Cys Thr Th Ι Thr His 540 Thr Phe Val Lys Al a 5 4 5 Leu Thr Thr Asp Glu 5 5 0 Lys Gin Ala Α 1 a Trp 555 Arg Phe Γ le Arq Ile 560 Asp Thr Ala Cys Val 565 Cys Val Leu Ser Arg 570 Lys A1 a Thr Arg Arg 575 Gly Ser Gly Thr Ser 580 &lt; 21 Ο &gt; 5 &lt; 211 &gt; 1065
&lt; 212 &gt; DNA &lt; 213 &gt; Synthetically &lt; 220 &gt; &Lt; 2 21 &gt; CDS &lt; 222 &gt; (1) . , (1 065) &lt; 223 &gt; KS1-DP-Hisrag-LL3 7-ELST-GCN P I ELST &lt; 400 &gt; 5 aLg cat acc cca gaa cac acc acc gcc gt gta cag ege ttt gtg gct. 48 Met 1 H 1 s Thr Pro Glu 5 His Ile Thr Ala Val 1 0 Val Gin Arg Phe Val 15 Ala gcg etc aal gcc ggc gaL ctg qae ggc atc gtc gcg ctq Ltt gcc gat 96 A1 a Leu Asn Ala 20 Gly Asp Leu Asp Gly 25 Tie Va 1 Ala Leu Phe 30 Ala Asp gac gcc acg gtg gaa gac ccc gtg ggt Lee gag ccc agg tcc ggc acg 144 Asp Ala Thr 35 Va 1 Glu Asp Pro Val 40 Gly Ser Glu Pro Arg 45 Ser Gl y Thr gct gcg att egt gag Ltt tac gcc aac Leg ctc dd ctg cct ttg gcg 192 Ala Ala 50 Ile Arg Glu Phe Tyr 5 5 Ala Asn Ser Leu T..VS 6 0 Leu Pro Leu Al a gt gag ctg acg cag gag gca ege gcg gcc gcc aac gaa gcg gcc r.tc 240 Val G1 u Leu their Gin Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe 49 • · φ &gt; * »» Φ φ »
65 70 get ctc acc gtc agc ttc gag did cag Ala Phe Thr Val Ser 85 Phe Glu Tyr Gin ccc atc gat cac ttt ege ttc aar. For example, each of his or her phe Arg phe Asn Gly 1 05 cg c gcc ztg ttt ggc gag aag aat att Arg ala leu 1 15 phe gly Glu lys asn 120 Ile atg tat caz cac cat. cac cat cac tet Met Tyr 13C H is His His Hi s His 135 His Serc ttc egg aa ate aaa gag a ag all Phe 145 Phe Arg Lys Ser Lys 150 Glu Lys Ile gtc cag aga atc aag gat ttt ttg egg Val Gl n Arg Ile Ty s 165 Asp Phe Leu Arg tcc Lee ggg gtt ccg ggt gtt ggt gtt Ser Ser Gly Val 1 80 Pro Gly Val Gly Val 1 85 gzL ggc. gtg cca ggt gzg gga gtg cca Val Gly Val 19 5 Pro Gly Val Gly Val 200 Pro qgc gtt ccg gga gtc gga qtt ccg gga G1 y Val 210 Pro Gly Val Gl y Val 215 Pro Gly gtz ccg ggt gta ggt ICC ggg cg L atg Val 225 Pro Gly Val Gl y Ser 230 G1 y Arg M et gag gag OLg c tg tet aag atc t.ae cac Glu Glu Leu Leu Ser 245 Lys Tie Tyr His ctg aa aag ctg att ggt gaa ege Lee T.eu ly s Lys Leu 260 Ile Gly Glu Arg Ser 26 5 cag gca ctg gaa gaa Qää aat get caa Gl n Ala Leu 275 Glu Glu Gl u Asn Ala 280 Gn ctg gaa gaa gaa atc gca caq ctg gaa Leu Glu 290 Glu Glu Ile Ala Gin 295 Leu Glu gtt gggg cct ggc gt ccL Val 305 Gly Val Pro Gly Val 310 Gl y Val Pro gga gtg cca ggc gtt ggz gzc ccg ggz Gly Val Pro Gly Val 325 Gly Val Pro Gly gtt ccg gga gta ggc gtg cct ggt 75 80 ggc Gly 90 ege Arg aag Lys acc Thr gLa Val gtt Val 95 geg Ala 288 gcc Ala ggc Gly aag I.ys gtg Val gtg Val 110 agc Ser atc Ile 336 cac His gca Ala zgc Cvs cag Gin 125 aLg Met gat Asp cct Pro 38 4 aga Arg gcc Ai a ggc Gly 140 ctg Leu ctg Leu ggt Gl v gaL Asp 432 ggg Gly Lys 155 gag Glu ttt Phe aaa Lys aga Arg pl Ile 160 480 aat Asn 17C ett Leu gta Val ccc Pro agg Arg aca Thr 175 gag Glu 528 cct Pro ggg Glygtg Val ggt Gly qtt Val 190 cct Pro ggt Gly 576 ggg Glygt L Val ggt Gly gta Val 205 ccg Pro ggt g: y gtg Val 624 g L a Va 'ggc Gly gtg Val 220 cct Pro ggt Gly qtt Val ggg Gly 672 33 a Lys cag Gin 235 ccg Leu gaa Glu ag ASp aaa Lys atc Ile 240 720 ctg Leu 250 gas Glu aac Asn qaa Glu at.L Ile get Ala 255 ege Arg 768 ggg Gly agc Ser cca Pro gaa Glu gac Asp 270 gaa Glu atz Ile 816 ctg Leu gaa Glu cag Gin gaa Glu 285 aac A sn Ala geg Ala 864 L ac Tyr qgc Gly Lee Ser 300 ggg Gly gtt Val ccg Pro ggz Gly 912 qqL Gly val Val 315 ggc G1 y gtg Va 1 cca Proggt Gl y gtg Val 320 9 60 gtg Val 330 ggc Gly gtt Val ccg Pro gga Gly gtc Val 335 gga Gly 10C 8 ggg att ccg ggt gt.a qgt tcc L 056 1065
50
Val Pro Gly Val Gly Val 340 Pro Gly Val Gly Val 345 Pro Gly Val Gly Ser 350 gga Gly act leads Ser 355 &lt; 210 &gt; δ &lt; 21Ί &gt; 3 55
&Lt; 212 &gt; PRT &lt; 213 &gt; Synthetically &lt; 400 &gt; 6
Met 1 His Thr Pro Glu 5 His Ile Thr Ala Val 1 0 Val Gin Arg Phe Val 15 Ala Ala Leu Asn AL o. 20 Gly Asp Leu Asp Gly 25 Tie Val Ala Leu Phe 30 Ala Asp ASp Ala Thr 35 Val Glu Asp Pro Val 40 Gly Ser Glu Pro Arg 45 Ser Gly Thr Ala Al a 50 Ile Arg Glu Phe Tyr 55 Al a Asn Ser Leu Lys 60 Leu Pro Leu Ala Val 65 Glu Leu Thr Gin G ^ u 70 Val Arg Ala Val Al a 75 Asn Glu Ala Ala Phe 80 Al a Phe Thr Val Ser 85 Phe Glu Tyr Gin Gly 9C Arg Lys Thr Val Va 1 95 Al a Pro I le Asp His 100 Phe Arg Phe Asn Gly 105 Ara Gly Lys Val Val 110 Ser Ile Arg Ala Leu 115 Phe Gly Glu Lys Asn 1 20 Ile Hrs Ala Cys G1 n 1 2 5 Met Asp Pro Met Tyr 130 His His His His 135 His Ser Arg Ala Gly 140 Leu Leu Gly Asp Phe 145 Phe Arg Lys Ser Lys 15C Glu Lys Ile Gly Lys 155 Glu Phe Lys Arg Ile 16C Val Gin Arg Ile L.ys 165 Asp Phe Leu Arg Asn 1 70 Leu Val Pro Arg Thr 175 Glu Ser Ser Gly Val 1 80 Pro Gly Val Gy Val 1.85 Pro Gly Val Gly Val 19C Pro Gly Val Gly Val 195 Pro c'-y Val Gly Val 200 Pro Gly Val Gly Val 205 Pr Gly Val Gly Val 210 Pro Gly Val Gly Val 215 Pro Gl y Val Gly Val 220 Pro Gly Val Gly Val 225 Pro Gly Valgy Ser 230 G 'v Arg vet Lys Gin 235 Leu Glu Asp Lys Tie 240 Glu g: u Leu Leu Ser 245 Lys Tie T vr H is Leu 250 Glu Asn Glu Ile Ala 255 Arg Leu Lys Lys Leu 260 Tie Gly Glu Arg Ser 265 Gl v Ser Pro Glu Asp 270 Glu Ile Gl ala Leu 275 Glu Glu Glu Asn Ala 280 Gin Leu Glu Gin Glu 285 Asn Ala Ala * · «· · · · * ·· • * ♦ *« * * * 51 • * • «• · • ♦« • · • • ♦ • · ♦ · »· • • · ♦ Val * * * * * * * * 9 9 9 9 9 9 9 9 9 9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Val Val Val Val Val Gly Val Pro Gly Val 320 Gly Val Pro Gly Val 325 Gly Val Pro Gly Val 330 Gly Val Pro Gly Val 335 Gly Val Pro Gly Val Gly Val Pro Gly Val 340 345 Gly Ihr Ser 355 &lt; 21 0 &gt; 7 &lt; 211 &gt; 1065 &lt; 212 &gt; DNA &lt; 213 &gt; Synthetically &lt; 220 &gt; &Lt; 221 &gt; CDS &lt; 222 &gt; (1) .. (1065) &lt; 223 &gt; KSI-DP-Histag-LL37 Elst GCN &lt; 400 &gt; 7 Gly Val P2-ELST Pro Gly Val350 Gly Ser atg cat acc cca gaacacac incc acc gcc gt a gta cag cgc ttt gtg gct 48 Met 1 HiS Thr Pro Glu 5 His Ile Thr Ala Val 10 V a 1 Gin Arg Phe Val 15 Al a gcg ctc aat gcc ggc gat ctg gac ggc a lc gtc gcg ctg ttt gcc gat 96 Ala Leu Asn Ala 20 Gly Asp Leu ASp Gly 25 Ile Val Ala Leu Phe 3 0 Ala Asp gac gcc acg gtg gaa gac ccc gtg ggt tcc gag ccc agg tcc ggt acg 144 Asp Ala Thr 3 5 Val Glu Asp Pro Val 40 Gl y Ser Glu Pro Arg 45 Ser Gly Thrgct gcg att. egt gag ttt tac gcc aac teg ctc aaa ctg cct teg gcg 192 Ala Ala 5 0 Ile Arg Glu Phe Tvr 55 Ala Asn Ser Leu Lys 60 Leu Pro Leu Ala gag ctg acg cag gag gta cgc gcg gtc gcc aac gaa gcg gcc ttc 240 Val 65 Glu Leu Thr Gin Glu 70 Val Arg Ala Val Ala 75 Asn Gl u Ala Al a Phe 80 gct ttc acc gtc agc Lee gag did cag ggc cgc aag acc gta gtt gcg 288 Ala Phe Thr Val Ser 8 5 Phe Glu Tyr Gin Gly 90 Arg Lys Thr Val Val 95 Ala ccc atc gat cac ttt cgc ttc aar. ggc gcc gggggggggggggggggggggggggggggg Ile Asp His 100 Phe Arg Phe Asn Gly 105 Ala Gly Lys Val Val 1 10 Ser Ile cgc gcc ctg ttt ggc gag aag aal att cac gca tgc cag atg gat cct 384 Arg Ala Leu 115 Phe Gly Glu Lys Asn 120 Ile His Ala Cys Gin 125 Met ASp Pro atg cat cat cac cat cac tet dg α gcc ggc ctg ctg ggt. gat 432 Met Tyr 130 His His His Kis His 135 His Ser Arg Ala Gly 140 Leu Leu Gly Asp ttc ttc egg clä-cl tet aaa gag aag att ggc aaa gag ttt aga att 480 Phe 145 Phe Arg Lys Ser Lys 150 Glu Lys Ile Gly Lys 155 Glu Phe Lys Arg Tie 160 528 • I * '* * * * * * * 52 gtc Val cag Gin aga Arg atc 11 e aag Lys 165 gat Asp tr.t Phe tt T.eu tcc Ser tcc Ser ggg Gly gtt Val 18C cg Pro ggt Gly gtt Val ggt Gly gLL Va] ggc Glygtg Val 195 cca Pro ggt Gly gtg Val gga Glygtg Val 200 ggg Gly gtt Val 210 ccg Pro gga Glygtc Val gga Gly gtt Val 215 ccq Pro gtt Val 225 ccg Pro ggt Gly gta Val ggt Gly tcc Ser 23 0 ggg Gly cg L Arg gag Glu qag Glu erg Leu ctg Leu tCL Ser 245 aag Lys atc 1 le tac Tyr ctg Leu 3. gL «, ys aag Lys Ctg Leu 260 alt Ile ggt Gly gaa Glu ege Arg gca Ala cag Gin ctg Leu 275 aaa lys gaa Glu aag Lys aac Asn geg Ala 280 ctg Leu aag Ly s 290 gag Glu aaa Lys atc Ile caa Gin gca Ala 295 ctg Leu gtt Val 305 ggr Glyg Val CcL Pro ggg Glygtg Val 310 ggt Gly gtt Val gga G'y gtg Va_ cca Pro ggg Gly gtt Val 325 gg- G'y gta Va_ ccg Pro gtt Val ccg Pro gga Gly gta Val 340 ggg Glygtg Val cct Pro ggg Gly gga Gly act Thr agt Ser 355 <210 &gt;; &lt; 211 &gt; &Lt; 212 &gt; &lt; 213 &gt; 8 355 PRT Synthet i; sch &lt; 4 0 0 &gt; 8 Met 1 H i 5 Thr Pro Glu 5 His Ile Thr Ala eu Asn Ala 20 Gly Asp Leu Asp Λ cp Ala Thr 35 Val Glu Asp Pro Val 40 egg aat Ctt gta ccc agg aca gag Arg Asn 170 Leu Val Pro Arg Thr 175 Glu gtt cct ggggt gtt cctggt Val 185 Pro Gly Va_ Gly Val 190 Pro Gly cca ggg ggt ggt gta ccg ggt gtg Pro Gly Val Gly Val 205 Pro Gly Valgga gta ggc gtg cct ggt gtt ggg Gly Val Gly Va: 220 Pro Gly-Val Glygg aaa cag ctg gaa gat aaa atc Met Lys Gin 23 5 Leu Glu Asp Lys Ile 24C cac ctg gaa aac gaa atc getge His Leu 250 Glu Asn Glu Ile Ala 255 Arg tcc ggg Lct cca gaa Q 3 C 33 3 atc Ser 265 G '. y Ser Pro Glu Asp 270 Avs Ile gcc ctg aaa gaa aag aat caa cag Ala Leu Lys Glu Lys 28 5 Asn Gin Gin 3 33 z tc tc ccgg g ccg ggt Lys Tyr Gly Ser 3 C0 Gly Va_ Pro Gly ccL ggt gtt ggc gtg cca ggt gtg Pro Gl y Val 345 Gly Va. Gly Val 330 Glyg Pro Gly Va.l 335 Gly gtt 999 gtt ccg ggt gta ggt tcc Val 345 Gly Val Pro Gly Val 3 50 Gly Ser
Ai 3 Val 10 Val Gin Arg Phe Val 1 5 Ala Gly 25 Ile Vael A-a Leu Phe 3 C Ala Asp Gly Ser Glu Pro Arg 45 Ser Gly Thr 576 624 672 720 768 816 864 912 960 10C 8 1056 1065 • * · '*' * 4 4 * * * • * * ι * * I * ♦ * * * Ψ V * * 4 «« * * «« 4 * · 53
Ala Ala 50 Ue Arg Glu Phe Tyr 5 5 Ala Asn Ser Leu Lys 60 Leu Pro Leu Ala Val 65 Glu Leu Gl Glu 70 Val Arg Ala val Ala 7 5 Asn Glu Ala Ala Phe 80 Ala Phe Thr Val Ser 85 Phe Glu Tyr Gl Gly 90 Arg Lys Thr Val Val 95 Ala Pro Ile Asp His 100 Phe Arg Phe Asn Gly 105 Ala Gly Lys Val Val 110 Ser Ile Arg Ala leu 115 Phe Gly Glu lys Asn 120 T le His Ala Cys Gin 125 Met Asp Pro Met Tyr 130 His His His His 135 His Ser Arg Ala Gl v 140 Leu Leu Gly Asp Phe 145 Phe Arg Lys Ser lys 1.50 G_U Lys Ile Gly Lys 1 55 Glu Phe Lys Arg Ile 160 Val Gin Arg Ile Lys 165 Asp Phe Leu Arg Asn 170 Leu Val Pro Arg Thr 175 Glu Ser Ser Gly Val 180 Pro Gly Val Gly Val 185 Pro Gly Val Gly Val 190 Pro Gly Val Gl y Val 195 Pro Gl y Val G .1 v Val 200 Pro Gly Val. Gly VAL 2 05 Pro Gly Val Gly Va 1 210 Pro Gly Val Gly Val 7.1 5 Pro Gl Val Giv Val 220 Pro Gly Val Giy Val 225 Pro Gly Val Gly Ser 230 Gly Arg Met Lys Gin 235 Leu Glu Asp Lys Ile 240 Glu Glu Leu Leu Ser 245 Lys Ile Tyr His Leu 250 Glu Asn Glu Ile Ala 255 Arg Leu Lys Lys Leu 260 y. le Gly Glu Arg Ser 2 6 5 Gly Ser Pro Glu Asp 270 Lys Ile Ala Gl π leu 275 -ys Gl u Lys Asn Ala 280 Ai a Leu Lys Glu Lys 285 Asn Gin Glu Leu Lys 290 Glu Lys I le Gin Ala 295 Leu Lys Tyr Gly Ser 3 0C Gly Val Pro Gly Val 305 Gly Val Pro Gly Val 31 C Gly Val Pro Gly Val 315 Gly Val Pro Gly Val 32C Gly Val Pro Giy Val 325 Gly Val Pro Gly Val 330 Gly Val Pro Gly Val 335 Cly Val Pro Gly Val 340 Gly Val Pro Gly Val 34 5 Gly Val Pro Gly Val 35C Glv Ser
Giy Thr Ser 355 &lt; 2'0 &gt; 9 &lt; 21J &gt; 99
&Lt; 712 &gt; DNA &lt; 213 &gt; Synthetically &lt; 220 &gt; 48
• * * 54 &lt; 2 21 &gt; CDS &lt; 222 &gt; (1) .. (99)
&Lt; 223 &gt; PI &lt; 400 &gt; 9 agc cca gaa gac gaa att. cag gca ctg gaa gaa gaa aat. gct caa egg
Ser Pro Glu Asp Glu Ile Gin Ala Leu Glu Glu Glu Asn Ala Gin len 1 5 10 15 gaa cag gaa aac gegg gt gaa gaa gaa acc gca cag ctg gaa tac
Glu Gin Glu Asn A_a Ala Leu Glu Glu Glu Ile Ala Gin Leu Glu Tyr 20 25 30 ggc C-ly
&Lt; 210 &gt; IC &lt; 211 &gt; 33
&Lt; 212 &gt; PRT &lt; 213 &gt; Synthetically &lt; 4 00 &gt; 10
Ser Pro Glu Asp Glu Ile Gin Ala Leu Glu Glu Glu Asn Ala Gin Ieu 15 10 15
Glu Gin Glu Asn Ala Ala Leu Glu Glu Glu Ile Ala Gin Leu Glu Tyr 20 25 30
Gly &lt; 210 &gt; 11 &lt; 211 &gt; 99
&Lt; 212 &gt; DNA &lt; 213 &gt; Synthetically &lt; 220 &gt; &Lt; 221 &gt; CDS &lt; 222 &gt; (1) .. (99) &lt; 223 &gt; P2 96 99 &lt; 400 &gt; 11 tet cca Ser Pro 1 gaa Glu gac Asp aaa Lys 5 atc Ile gca Ala cag G_n ctg Leu aaa Lys 10 gaa Glu aag Lys aac Asn geg Ala gcc Ala 15 ctg Leu aaa gaa Lys Glu aag Lys aat Asn 20 caa Gin cag Gin ctg Leu aag Lys gag Glu 2 5 ädd Lys atc I le caa Gin gca Ala ctg Leu 30 aaa lys did Tyr ggc Gly &lt; 210 &gt; 12 &lt; 211 &gt; 33 &lt; 21.2 &gt; PRT &lt; 213 &gt; Synthetically &lt; 400 &gt; 12 Ser Pro Glu Asp Lys Tie Ala Gin Lei: Lys Glu Lys Asn Ala Ala Leu l 5 10 15 4 8 96 I.ys Glu Lys Asn Gin Gin Leu Lvs Glu Lvs Ile Gin A * a Leu Lys Tyr 99 55 2ο 30 20 Gly &lt; 210 &gt; 13 &lt; 21 1 &gt; 99 &lt; 21 2 &gt; DNA &lt; 213 &gt; Synthetically &lt; 220 &gt; &lt; 221 &gt; CDS &lt; 222 &gt; (1) .. (99) &lt; 223 &gt; GCN &lt; 400 &gt; 13 cqt atg aaa cag ctg gaa gat aaa atc gag qag ctg ctg tet aag atc Arg 1 Met Lys Gin Leu 5 Glu Asp Lys Ile Glu 1 0 Glu Leu Leu Ser Lys 15 Ile tac cac ctg gaa aac gaa att get ege ctg aaa ag ctg at t ggt gaa Tyr his Leu Glu 20 Asn Glu 1! e Al.a Arg 25 Leu Lys Ly 3 Leu T 1 e 30 Gly Glu cgc Arg 48 96 99 &lt; 210 &gt; 14 &lt; 211 &gt; 33 &lt; 212 &gt; PRT &lt; 21 3 &gt; Synthetically &lt; 00 &gt; 14 Arg Met Lys G1n Leu Gl u Asp 1 5 Tyr H: s I.eu Glu Asn Glu Tie 2C Arg &lt; 210 &gt; 15 &lt; 211 &gt; 111 &lt; 212 &gt; DNA &lt; 21 3 &gt; Synthetically &lt; 220 &gt; &Lt; 221 &gt; CDS &lt; 222 &gt; ¢ 1). (111) &lt; 223 &gt; Li 3 7 &lt; 400 &gt; 15 ctg ctg ggt gat. ttc ttc egg Leu Leu Gly Asp Phe Phe Arg 1 5 ttt aaa aga att. gtc Cag aga Phe Lys Arg Ile Val Gln Arg 20 atc aag Ile: .. ys 25 25
IC 15 3 0 aaa qag aag att ggc aaa gag Lys Glu l.ys iie Gly Lys Glu 10 15 gaL ctt ttg egg aat Asp Phe:, eu Arg Asn 30: tt gta ccc agg aca gag tcc Pro Arg your G '. u Ser 3 5 48 96 1 1 1 * 4 • • • • * &gt; * * ♦ * * k · 56 &lt; 210 &gt; 16 &lt; 211 &gt; 37
&Lt; 212 &gt; RT <213> Synthetically &lt; 400 &gt; 16
Leu Leu 1 Gly Asp Phe 5 Phe Arg Lys Ser Lys 10 Glu Lys Ile Gly Lys 15 Glu Phe Lys Arg Ile 20 Val Gin Arg Ile Lys 25 Asp Phe Leu Arg Asn 30 Leu Val Pro Arg Thr 3 5 Glu Ser &lt; 2 10 &gt; &lt; 211 &gt; &Lt; 212 &gt; &Lt; 213 &gt; 17 150 DNA Synthesis &lt; 220 &gt; &Lt; 221 &gt; &Lt; 222 &gt; &Lt; 223 &gt; CDS (1). ELST. {15 0) &lt; 400 &gt; gtt ccg Val Pro 1 17 ggt Gly gtt Val ggt Gly 5 gtt Va 1 cct Pro ggg Glygtg Val ggt Gly 10 gtt Val cct Proggt Gly gtt Val ggc Gly 1 o gtg Val 48 cca ggt Pro Gly gtg Val gga Gly 20 gzq Val cc Pro gqc Gly gtt Val ggt Gly 25 gta Val ccg Proggt Gly gtg Val ggc Gl y 30 gtt Val ccg Pro 96 gga gt Gly Glyg g Gly 3 5 gtt Val ccg Pro gga Gly gLa Val ggg Gly Gly Val 40 cct Pro Gly gtt Val ggg Gly 43 gtt Val ccq Pro ggt Gly 144 gLa ggt Val Glv 50 150 <210>. &Lt; 211 &gt; &Lt; 212 &gt; &Lt; 213 &gt; 18 50 PRT Synthetic &lt; 4 0C &gt; 18 Va 'Pro 1 Gly Vdl Gly 5 Val Pro Gly Val Glv 10 Val Pro Glv Val Glv 15 Val Pro Glv Val Gly 20 Val Pro Gly Val Gly 25 Val Pro Gly Val Glv 30 Val Pro G: y Val Gly 3 5 Val Pro Gly Val Gly 40 Val Pro Gly Val Gly 45 Val Pro Gly Val Glv 50 &lt; 2.1C &gt; &Lt; 211 &gt; &Lt; 212 &gt; &lt; 2 13 &gt; &gt; 19 1140 DKA Synthetic «« * * * * * * * * * * * * * * * * * I - * * ** 57 <220>. &Lt; 221 &gt; CDS &lt; 222 &gt; (1) . , (1.140) &lt; 223 &gt; KST DP-Hcstag-ELST-GCN-P2-ELST-AEA &lt; 400 &gt; 19 atq cat acc cca gaa cac atc acc gcc gt gta cag cgc ttt gtg gct 48 Met 1 His Thr Pro Glu 5 Hi s Tie Thr Ala Val 10 va 1 G 'n Arg Phe Val 15 Ala gcg ctc aat gcc ggc gat Ctg gac ggc atc gtc gcg Ctg L t L gcc gat 96 Ala Leu Asn Ala 20 Gly Asp Leu Asp Gl v 25 Ile Val Ala Leu Phe 3C Ala Asp gac gcc aeg gta gac ccc gtg ggt t.cc gag ccc agg tcc ggt aeg 144 Asp Ala Thr 35 Val Glu Asp Pro Val 40 Gly Ser Glu Pro Arg 45 Ser Gly ThrgcT gcg atc cgc gag ttt Lac gcc aac teg ctc aaa ctg cct ttg gcg 1 92 Ala Ala 50 Ile Arg Glu Phe Tyr 55 Ala Asn Ser Leu Lys 60 Leu Pro Leu Ala gt gag ctg aeg cag gag gra cgc gcg gtc gcc aac gaa gcg gcc ttc 240 Val 6 5 Glu Leu Thr Gin Glu 70 Val Arg Ala Val Al.a 75 Asn Glu Ala Ala Phe 80 gct tLC acc gtc agc ttc gag did cag ggc cgc aag acc gta gtt gcg 288 Ala Phe Thr Val Ser 85 Phe Glu Tyr Gin Gl v 90 'Arg Lys Thr Val Val 95 Ala ccc atc gar cac ttL cgc ttc aat: ggc gcc ggc agag gt.g gtg agc ot C 336 parts Asp His 100 Phe Arg Phe Asn Gly 1 05 Ala Gly Lvs Val Val 110 Ser Tie cgc gcc LLg ttt ggc gag ci ciCJ s al L att cac gca lgc cag atg gat cct 384 Arg Ala Leu 115 Phe Gly Glu Lys Asr. 12C Ile His Ala Cys Gin 125 Met Asp. Prosp. Lat cat cac cat cac cat cac tc I. aga gcc ggc gtc ccg ggt gtt 432 MeL Tyr 130 His His His Hin Hrs 135 His Ser Arg Ala Gly 1 40 Val Pro Gly Val gt- - cct ggc grg ggt gtt. cc Lgt gtt. gggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggghgggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggg 528 va j Pro Giy Val Gly 1 65 Val Pro Gly Va 1 Gly 170 Val Pro Gly Val Gly 175 Val ccg gga gta ggc gtg cct ggt ggg qtL ccg ggt gta ggt. tcc ggg 576 Pro G. 'y y Val Gl v 180 Val Pro Gly Val Gly 185 Va 1 Pro Gly Va 1 Gly 19C Ser Glyteg aaa cag ctg gaa gat aaa atc gag gag CLg ctg tet aag atc 624 Arg Mer. lys 195 Gin Leu Glu Asp Lys 200 Ile Glu Gl u Leu Leu 205 Ser T.ys Ile Lac cac ctg gaa a ac gaa att gct cgc cLg aag ctg att ggt gaa 672 Tyr His 21 C Leu Glu Asn G 1, u Ile 215 Λ1 a Arg Leu ^ ys lvs 220 Leu Tie Gly Glu cgc tcc ggg rer cca gaa gac cl £ 3 5 atc gca cag ctg aaa gaa aag aac 720 Arg 225 Ser Gly Ser Pro G_u 230 Asp i.ys Tie Ala Gin 235 T.eu Ty s Glu Lys Asn 240 gcc ctg aaa CJitl ci aag aat caa cag ctg aag gag aaa atc caa gca 768 58
Ala Ala Leu Lys Glu 245 Lys Asn Gin Gin Leu 250 Lys Glu Lys Ile Gin 255 Ala ctg Leu aaa Lys Lat Tyr ggc Gly 260 tcc Ser ggg Gly gcL Val ccg Pro ggt Gly 2 65 gtt Val ggt Gly gtt Val ccL Pro ggc Gly 270 gtg Val ggt Gly 816 gtc Val cct Proggt Gly 275 gtt Val ggc Glygtg Val cca Pro ggt. Gly 280 gtg Val gga Glygtg Val cca Pro ggc Gly 28 5 gtt Val ggt Gly gta Val 864 ccg Pro ggt Gly 290 gtg Val ggc Gly gtt Val ccg Pro gga Gly 295 gtc Val gga Gly gtt Val ccg Pro gga Gly 300 gta Val ggg Glygtg Val cct Pro 912 ggl Gly 305 gtt Val ggg Gly gtt Val ccg Pro ggt Gly 310 gta Val gt. Gly tcc Ser ggg Gly 3 Lys 315 tgg Trp gct Ala gcc Ala atc Ile gaa Glu 320 960 gaa Glu gaa Glu gca Ala gcc Ala gct Ala 325 att Ile aaa Lys gaa Glu gag Glu gca A ± a 330 gca Ala gcg Ala atc Ile gaa Glu gaa Glu 335 33 5 Lys 1008 gcc Ala gca Ala gcc Ala atc T 1 e 340 3dd Lys gag Glu gaa G1 u gcc Al a gca Ala 345 gct. Ala atc le gag Glu gaa Glu cl <3 ö Lys 350 tgg Trp gct Ala 1C56 gcg Ala att Ile gaa Glu 355 qaq Glu gaa Glu gct Ala gcg Ala gca Ala 36C ata Ile CJcaci Glu gag Glu aaa Lys tgg Trp 365 gct Ala gct Ala atc le 1104 aaa Lys gaa Glu 370 <5.3.3. T, ys gcg Ala gct Ala gc a Al a a cc al 375 aa Lys tcc Ser gga Gly ach Thr agt Ser 380 1140 &lt; 2 10 &gt; &Lt; 211 &gt; &Lt; 212 &gt; &Lt; 213 &gt; 20 3 SO PRT SyntheLi sch &lt; 4 00 &gt; 20 Met 1 His Thr Pro Glu 5 His Ile Thr Ala Val 1 0 Val Gin Arg Phe Val 15 Ala Ala Leu ASn Ala 20 Gly Asp I.eu Asp Gly 25 T Le Val Ala ΐeu Phe 30 Ala Asp ASp A.! a Thr 35 Val Glu Asp Pro Val 40 GIv Ser Glu Pro Arg 45 Ser Gly Thr Al a Ala 5C Ile Arg Glu Phe Tyr 5 5 Ala Asn Ser Leu Lys 60 Leu Pro Leu Ala Val 65 Glu Leu Thr Gin Glu 70 Val Arg Ala Val Ala 75 Asn Glu Ala Ala Phe 80 Ala Phe Thr Val Ser 85 Phe Glu Tyr Gin Gly 90 Arg Lys Thr Val Val 95 Ala Pro Ile Asp His 100 Phe Arg Phe Asn GY 105 Ala Gly Lys Va 1 Val 1 1 0 Ser I le Arg Ala Leu 115 Phe Gly Glu Lys Asn 120 T 1 e Hi s Ala Cys Gl n 125 Met Asp Pro Met Tyr His H is His His His; s Ser Arg Ala Gly Val Pro Gly Val 59 130 135 140 Gly 145 Val Pro Gly Val Gly 150 Val Pro Gly Val Gly 155 Val Pro Gly Val Gly 1 60 Val Pro Gly Val Gly 16 5 Val Pro Gly Val Glv 170 Val Pro Gly Val Gly 175 Val Pro Gly Val Gly 180 Va 1. Pro Gly Val Gly 185 Val Pro Gly Va _ Gly 19C Ser Gly Arg Met Lys 195 Gin Leu G1 u Asp Lys 200 Tie Glu Glu Leu Leu 205 Ser Hys I le Tyr His 210 Leu Glu Asn Glu Ile 215 Ala Arg Leu Lys Lys 220 Leu Ile Gly Glu Arg 225 Ser Gly Ser Pro Glu 230 Asp Lys Ile Ala Gin 23 5 Leu Lys Glu Lys Asn 240 Ala Ala Leu Lys Glu 245 Lys Asn G_n G_n Leu 250 Lys Glu Lys Ile Gin 255 Ala Leu Lys Tyr Gly 260 Ser Gly Val Pro Gly 265 Val Gly Val Pro Gly 270 Val Gly Val Pro Glv 275 Val Gly Val Pro Glv 280 Va 1 Gly Va 1 Pro Gly 285 Val Gly Val Pro Gly 29C Gly Val Pro Gly 295 Val Gly Val Pro Gly 300 Val Gly Val Pro Gly 305 Val G1 v Val Pro Gly 310 Val Gl y Ser Gly Lys 315 Trp Ala Al a T 1 e Glu 320 Glu Glu Al a A 1 a Ala 325 il Lys Glu Glu Ala 33 0 Ala Ala I le Glu G "1 u 335 Lys Ala Ala Ala Ile 340 Lys Glu Glu Ala Ala 345 Ala Tie Gl u Glu Lys 35C Trp Ala Ala I le Glu 355 Glu Glu Ala Ala 7ila 360 Ile Glu Glu Lys Trp 365 Ala Ala Ile Lys Glu 370 Lys Ala Ala Ala Ile 375 Lys Ser Gly Thr Ser 380 &lt; 210 &gt; &Lt; 211 &gt; &Lt; 212 &gt; &Lt; 213 &gt; 21,375 DNA Synthet.i; sh &lt; 220 &gt; &lt; 221 &gt; &lt; 2 22 &gt; &Lt; 223 &gt; CLIS &lt; 1) · K5I. (375) &lt; 400 &gt; cat acc His Thr 1 21 cca Pro gaa Glu cac Hi s 5 atc Ile acc Thr gcc Ala gtg Val gta Val 10 cag Gin cgc Arg Ltl Fhe gtg V ac g Ala 15 gcg Ala ctc Leu aa t A sn gct Ala ggr, Gly 2 0 gat Asp ctg Leu gac Asp ggc Glycet le 25 gtc Val gcg A ^ a ctg Leu ttt Phe gcc Aid 30 even Asp gac Asp gcc ci cg gt gaa gac ccc gtggt tcc gag ccc agg tcc ggt ocg gct 48 98 144 60
Ala Thr Val Glu Asp Pro Val Gly Ser Gl u Pro Arg Ser Gly Thr Ala 35 40 45 contra ate gag ttt tac gcc aac teg ctc aa a ctg cct ttg against 192 Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala Val 50 55 60 gag ctq aeg cag gag gra g n gtc gcc aac ga a g g c ttc get 240 Glu Leu Thr Gin Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe Ala 65 70 75 8C tr.c acc gtc agc ttc gag tat cag ggc ege aag acc gta gtlg ccc 288 Phe Thr Val Ser Phe Glu Tyr Gin Gly Arg Lys Thr Val Val Ala Pro 85 90 95 aic gat cac ttt ege ttc aat ggc gcc ggc agag gtg agg acce 336 Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile Arg 100 105 110 gcc ttg ttt. ggc gag aag aat atr. cac gca tgc cag atg 37 5 Ala Leu Phe Gl γ Glu Lys Asn Ile His Ala Cvs Gin Met 115 120 125 &lt; 210 &gt; 22 &lt; 211 &gt; 125 &lt; 212 &gt; PRT &lt; 2 13 &gt;Synr.het!; sch &lt; 400 &gt; 22 His Thr Pro Glu His Ile Thr Ala Val Val Gin Arg Phe Val Ala Ala 1 5 10 15 Leu Asn Ala Gly Aspu Asp Gly Ile Val Ala Leu Phe Ala Asp Asp 20 25 30 Ala Thr Va: Glu Asp Pro Va 1 Gly Ser Glu Pro Arg Ser Gly Thr Al a 35 40 45 Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala Val 50 55 60 Glu Leu Thr Gin Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe Ala 65 70 75 80 Phe Thr Val Ser Phe Glu Tyr Gin Gly Arg Lys Th r Val Val Ala Pro 85 90 95 Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile Arg 100 105 110 Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gin Met 115 120 125 &lt; 210 &gt; 23 &lt; 2ll &gt; 669 &lt; 212 &gt; DNA &lt; 213 &gt; Synthetic &lt; 220 &gt; &lt; 2 2 1 &gt; CDS &lt; 222 &gt; (1) . (669) &lt; 2 2 3 &gt; mNGF &lt; 4 0 0 &gt; 23 gaa ccg did act. Glu Pro Tyr Thr Asp Ser Asn Va 1 Pro Glu Gly Asp Ser Val Pro G 1 u 61 1 5 10 15 gcc cat tqg aca aaa ctg caa cac agc ctg gat aca gca ctg egt egt 96 Ala Hrs Trp Thr Lys Lei Gin His Ser Leu Asp Thr Ala Leu Arg Arg 20 25 30 gcc egt -ca gca cca aca get cct. att get gcc cg L gtt act ggt cag 144 Ala Arg Ser Ala Pro Thr Ala Pro Ile Ala Ala Arg Val Thr Gly Gin 35 40 45 Acc ege aacat aeg gtg cct egt ctg etc aaa aaa ege ege ctg 1 92 Thr Arg Asn Ile Thr Val Asp Pro Arg Leu Phe Lys I, Arg Arg Leu 50 55 60 cat tcc cct egt gtt ctg ttt agc aca caa cct ccg cca aca tca agc 240 His Ser Pro Arg Val Leu Phe Ser Thr Gin Pro Pro Pro Thr Ser Ser 65 70 75 80 gat aca ctg gac ctg gac ttt caa gca cat gga acc att ccg ttc aat 288 Asp Thr Leu Asp Leu Asp Phe Gin Ala His Gly Thr Ile Pro Phe Asn 85 90 95 egt. acc cat egt. tca aaa ege tca agc acc cat ccg gtg ttt cat atg 33 6 Arg Thr His Arg Ser Lys Arg Ser Ser Thr His Pro val Phe Hi s Met 100 1 05 110 ggg gaa t t t t g ttt gac agc gtg tt gtc tgg gtt ggg gat aaa 384 Gly Glu Phe Ser Val Cys Asp Ser Val Ser Val Trp Val Gly Asp Lys 115 120 125 Acc aca pour acc gat a Le aaa ggc aaa gaa g Lg acc g Le ctg gcc gaa 432 Thr Th r Al a Thr Asp T 1 e Lys Gly Lys Glu Val Thr Val Leu Ala Glu 1 3 0 135 140 gtc aat aac aac agc gtc ttt egt caa tat ttc ttc gaa acc 333 480 Val Asn Ile Asn Asn Ser Val Phe Arg Gin Tyr Phe Phe Glu Thr lys 145 150 155 160 tgc egt gcc tca aaL c et gLa gaa agc ggg tgt cg Leagues Lea 528 Cy s Arg Ala Ser Asn Pro Val Glu Ser Gly Cys Arg Gly Ile Asp Ser 165 170 175 33 3 cat tgg aac teg tat tgt acc acc act cac acc ttc gtt aaa gcc 576 Lys H: s T rp Asn Ser Tyr Cys Thr Thr Thr His Thr Phe Val Lys Ala 1 80 185 190 ctg act acc gac gag aa a caa get get tgg ege ttt atc egt atc gat 624 '..eu Thr Thr Asp Glu Lys Gin Ala Ala Trp Arg Phe Ile Arg Ile Asp 195 200 205 acc tt tgt gtg tgt gtc ctg tcc egt aaa gca aca egt egt ggt 669 'Al a Cys Val Cys Val Leu Ser Arg Lys Ala Thr Arg Arg Gly 2] 0 215 220 &lt; 2 1 0 &gt; 24 &lt; 211 &gt; 223 &lt; 21 2 &gt; PRT &lt; 21 3 &gt; Syntl netic &lt; 4 0 0 &gt; 24 Gl u Pro Tyr Thr Asp Ser-Asn Val Pro Glu Gly Asp Ser Val Pro Glu 1 5 1 0 15 Al a His Trp Thr Lys Leu Gin His Ser Leu Asp Thr Ala Leu Arg Arg 20 25 30 62 Ala Arg Ser 3 5 Ala Pro Thr Ala Pro 40 Thr Arg 50 Asn 1 le Thr Val Asp 55 Pro Hi s 65 Ser Pro Arg Val Leu 70 Phe Ser Asp Thr Leu Asp Leu 85 Asp Phe Gin Arg Thr His Arg 100 Ser Lys Arg Ser Gly Gl u Phe 115 Ser Val Cys Asp Ser 120 Thr Thr 130 Ala Thr Asp Ile Lys 135 Gly Val 145 Asn Ile Asn Asn Ser 150 Val Phe Cys Arg Ala Ser Asn 165 Pro Val Glu Lys His Trp Asn 180 Ser Tyr Cys Thr Leu Thr Thr 195 Asp Glu lys Gl n Ala 200 Thr Ala 210 &lt; 210 &gt; &lt; 211 &gt; &Lt; 212 &gt; &Lt; 213 &gt; &Lt; 220 &gt; &Lt; 221 &gt; &lt; 2 2 2 &gt; &Lt; 223 &gt; &Lt; 4 C 0 &gt; Cys Val Cys 25 186 DNA Synthetic CDS (1) .. (186) AEA 25 Val Leu 215 Seraag tgg gct gcc atc gaa gaa gaa Lys 1 Trp Ala Ala Ile 5 Glu Glu Glu gca gcg £ LC gaa gaa 3 3 3 gcc ga Ala Al a Ile Glu 20 Glu Lys A_a Ala atc gag gaa aaa tgg gct gc.g atr Tie Glu Glu 35 lys Trp Ala A- 3. Ile 40 gag aaa tgg gct gct atc aaa gaa Glu Lys 50 Trp Ala Al a Γ le I iV 5 55 Glu
Ile Ala Ala Arg Val 45 Thr Gly Gin Arg Leu Phe Lys 60 Lys Arg Arg Leu Thr Gin Pro 75 P ro Pro Thr Ser Ser 80 Ala His 90 Gly Thr Ile Pro Phe 9 5 Asn Ser 105 Thr His Pro Va_ Phe 110 His Met Val Ser Val Trp Val 125 Gly Asp Lys Lys Glu Val Thr 140 Val Leu Ala Glu Arg Gin Tyr 15 5 Phe Phe Glu Thr Lys 1 60 Ser Gly 170 Cys Arg Gly Tie Asp 175 Ser Thr 185 Thr His Thr Phe Val 190 Lys Ala Ala Trp Arg Phe lle 205 Arg Ile Asp Arg Lys Ala Thr 22C Arg Arg Glygca Ala gcc Ala 10 gcL Ala aLL Ile aaa Lys CJaca G_U gag Glu 15 gca Ala gcc Ala 25 atc Ile aaa Lys gag Glu gaa Glu gcc A ~ a 30 gca Ala gct Ala gaa Glu gag Glu gaa Glu gct Ala gcg Ala 4 5 gca Ala ata Ile gaa Glu aaa Lys gcg Al a gct Ala gca Ala 60 atc Ile aaa Lys 48 96 144 &lt; 210 &gt; 26 &lt; 211 &gt; 62 &lt; 212 &gt; PRT &lt; 213 &gt; Synthetically 186 63 &lt; 400 &gt; 26
Lys Trp Ala Ala Ile Glu Glu Glu Ala Ala Ala Ile Lys Glu Glu Ala 15 10 15
Ala Ala Ile Glu Glu Lys Ala Ala Ala Ile Lys Glu Glu Ala Ala Ala 20 ~ 25 30
Ile Glu Glu lys Trp Ala Ala Tie Glu Glu Glu Ala Ala Ala Ile Glu 35 40 45
Glu Lys Trp Ala Ala Ile Lys Glu Lys Ala Ala Ala Ile Lys 50 55 60 &lt; 210 &gt; 27 &lt; 211 &gt; 99
&Lt; 212 &gt; DNA &lt; 213 &gt; Synthetically &lt; 2 2 0 &gt; &lt; 2 21 &gt; &gt; CDS &lt; 222 &gt; (1) .. (99) &lt; 223 &gt; P3 &lt; 40G &gt; 27cc. Ser 1 ccg Pro gaa Glu gat Asp gag Glu 5 atc Ile cag Gin caa Gin ctg Leu gaa Glu 10 gaa Glu gaa Glu atc Ile gct Ala cag Gin 15 ctg Leu 48 gaa Glu cag Gin aaa Lys aac Asn 20 gca Ala gcg Ala ctg Leu aaa Lys gag Glu 25 aaa Lys aac Asn cag Gin gcg Ala ctg Leu 30 aaa Lys tac Tyr 96 ggt Gly 99 &lt; 210 &gt; &lt; 211 &gt; &lt; 2 12 &gt; &lt; 2 13 &gt; &gt; 28 33 PRT Synthetic &lt; 4 00 &gt; 28 Ser 1 Pro Glu ASp Glu 5 Glu Glu Ile Ala C-ln 15 Leu Glu Gin Lys Asn 2C Ala Ala Leu Lys Glu 25 Lys Asn Gin Ala Leu 30 Lys Tyr
Glv &lt; 210 &gt; 29 &lt; 211 &gt; 99
&Lt; 212 &gt; DNA &lt; 213 &gt; Synthetically &lt; 220 &gt; &Lt; 221 &gt; CDS &lt; 2 22 &gt; (1) .. (99) &lt; 2 2 3 &gt; P4 &lt; 400 &gt; 29 agc ccg gaa gat aaa att gct cag ctg aaa caa aaa atc caa gcg ctg Ser Pro Glu Asp Lys Ile Ala Gin Leu Lys Gin Lys Gin Ala Leu 48 * ». , »
1 5 aaa cag gaa aac cag cag ctg qaa gag Lys Gin Glu Asn Gin Gin Leu Glu Glu 20 25 ggt
Gly 15 aac gCc qca ctg gaa did 96
Asn Aia Ala LeLi g: u Tyr 30 &lt; 210 &gt; 30 &lt; 211 &gt; 33
&Lt; 212 &gt; PRT &lt; 213 &gt; Synthetically &lt; 400 &gt; 30 Ser Pro Glu Asp Lys I Al Al Gln Leu 1 5 Lys Gin Glu Asn Gin Gin Leu Glu Glu 20 25 Gly &lt; 21 0 &gt; 31 &lt; 211 &gt; 99 &lt; 21 2 &gt; DNA &lt; 213 &gt; Synthetically &lt; 220 &gt; &lt; 221 &gt; CDS &lt; 222 &gt; (1) -. (99) &lt; 223 &gt; P5 &lt; 4 00 &gt; 31 tct cct gaq gac qaa aac gca gct ctg Ser Pro Glu Asp Glu Asn Ala Ala Leu 1 5 33d 083 aag aac ace ga ctg aas gaa Lys Gin Lys Asn Ala Ala Leu Lys Glu 20 25 ggc Gly &lt; 21 0 &gt; 3 2 &lt; 211 &gt; 33 &lt; 2 12 &gt; PRT &lt; 213 &gt; Synthetically &lt; 4Q0 &gt; 32 Ser Pro i Glu Asp Glu Ao n Ala Ala Leu 1 5 Lys Gin i Lys Asn Al a Ala Leu iys Glu 20 25 Gly
Gin I &gt; ys Ile Gin Ala Leu 15
Asn Ala Ala Leu Glu Tyr 30 gag aaa att gca caa ctg 18
Glu Lys Ile Ala Gin Leu 15 atL caa gca ctg gaa did 95
Ile Gin Ala Leu Glu Tyr 3 0
Glu Lys Ile Ala Gl.n Leu 15
Ile Gin Ala Leu Glu Tyr 3 0 3 3 99 &lt; 21Q &gt; &lt; 211 &gt;
65
&Lt; 212 &gt; DNA &lt; 213 &gt; Synthetically &lt; 22C &gt; &Lt; 221 &gt; CDS &lt; 222 &gt; (1) .. (99) &lt; 223 &gt; P6 &lt; 400 &gt; 33 agc Ser 1 ccg Pro gaa Glu gat Asp S. öci Lys 5 aac Asn gcc Ala gct Ala ctg Leu aaa Lys 10 gag Glu gaa Glu atc Ile cag Gin gcg Ala 15 ctg Leu gaa Glu gaa Glu gaa Glu aac Asn 20 cag Eq n gct Al a ctg Leu gaa Glu gag Glu 25 aa Lys atc Ile gca Ala cag Gin ctg Leu 30 aaa Lys did Tyr ggt Gly &lt; 21 0 &gt; &lt; 2 11 &gt; &lt; 2 1 2 &gt; &lt; 2 13 &gt; &gt; 34 33 PRT Synthesis &lt; 40 0 &gt; 34 Ser 1 Pro Glu Asp Lys 5 Asn Al a Al Leu Lys 1 0 Glu Glu Ile Gin Ala 15 Leu 48 96 99
Glu Glu Glu Asn Gin Ala Leu Glu Glu Lys Ile Ala Gin Leu Lys Tyr 20 25 30
Gly &lt; 210 &gt; 35 &lt; 211 &gt; 99
&Lt; 212 &gt; DNA &lt; 213 &gt; Synthetically &lt; 2 2 0 &gt; &lt; 2 2 1 &gt; CDS &lt; 2 22 &gt; (1) . , (99) &lt; 2 23 &gt; P7 &lt; 400 &gt; 35 tcc ccg gag gag gag otc cag gcg ctg gaa gaa aag aac gcc cag ctg
Ser Pro Glu Asp Glu Ile Gin Ala Leu Glu Glu Lys Asn Ala Gin Leu 15 IC 15 aag cag gaa att gcg gca ctg gaa gag aag aac cag gcc clg aag Lac
Lys Gin Glu Ile Ala Ala Leu Glu Glu Lys Asn Gin Ala Leu Lys Tyr 20 25 30 gat
Gly &lt; 21C &gt; 36 &lt; 211 &gt; 33
&Lt; 212 &gt; PRT &lt; 213 &gt; Synthetically &lt; 400 &gt; 36 48 96 99 66
Ser 1 Pro Gl u Asp Glu 5 T 1 e Gin Ala Leu Glu 1 0 Glu Lys Asn Ala Gln 15 Leu Lys Gin Glu Ile 20 Ala Ala Leu Glu Glu 25 Lys Asn Gin Ala Leu 30 Lys Tyr
Gly &lt; 210 &gt; 37 &lt; 211 &gt; 99
&Lt; 212 &gt; DNA &lt; 213 &gt; Synthetically &lt; 220 &gt; &Lt; 2 21 &gt; CDS &lt; 222 &gt; (1). (99) &lt; 723 &gt; P8 &lt; 400 &gt; 37
Lee Ser 1 ccg Pro gaa Glu gac Asp aaa Lys 5 atc Ile get Ala cag Gin ctg Leu aaa Lys 10 gaa Glu gaa C-lu aac Asn cag Gin cag Gin 15 ctg Leu 48 gaa Glu caa Gin aag Lys att Ile 20 cag Gin gcc Ala ctg Leu aag Lys gag Glu 25 gaa Glu aac Asn gca Ala get Ala ctg Leu 30 gaa Glu tac Tyr 96 qgc 99
Gly &lt; 210 &gt; 38 &lt; 211 &gt; 33
&Lt; 212 &gt; PRT &lt; 213 &gt; Synthetically &lt; 400 &gt; 38
Ser Pro Glu Asp Lys Ile Ala Gin Leu Lys Glu Glu Asn Gin Gin Leu 15 10 15
Glu Gin Lys Tie Gin Ala Leu Lys Glu Glu Asn Ala Ala Leu Glu Tyr 20 25 30
Gl y

权利要求:
Claims (19)
[1]
* «Ι a» * »· 1 Patent Attorneys Dipl.-Ing. Helmut Hübscher Dipl.-Ing. Karl Winfried Hellmich Spittelwiese 7, A 4020 Linz (38589) HEL Patent claims: 1. Polypepidmaterial containing at least one elastin-like segment and at least two segments to form double helices.
[2]
2. Polypepidmaterial according to claim 1, characterized in that it also contains at least one or a combination of several, usually 2 to 10, functional polypeptide domains, which are selected from, but not limited to: growth factors, molecules for cell adhesion, molecule chemotaxis , Ligand receptors, microbe-rejecting peptides, factors for reprogramming, factors for cell differentiation, cytotoxic or cytostatic molecules, such as epidermal growth factor (EGF), fibroblast growth factors, neuronal growth factors (NGF), especially SEQ ID NO: 24, microbe-rejecting peptides such as cathelizidines and defensins; especially the Kathelizidin LL-37, especially SEQ ID NO: 16, wherein this functional DNA polypeptide domain is covalently bound to the polypeptide material.
[3]
Polypeptide material according to claim 1, characterized in that it also contains at least one or a combination of several, usually 2 to 10, functional polypeptide domains selected from, but not limited to: growth factors, molecules for cell adhesion, molecule chemotaxis , Ligand receptors, microbe-rejecting peptides, factors for reprogramming, factors for cell differentiation, cytotoxic or cytostatic molecules, such as epidermal growth factor (EGF), fibroblast growth factors, neuronal growth factors (NGF), especially SEQ ID NO: 24, microbe-rejecting peptides such as cathelizidines and defensins; especially the Kathelizidin LL-37, especially SEQ ID NO: 16, wherein the functional DNA Polypeptiddomäne is connected to at least one segment to form double helices, the functional domain on * * · * ··· II t φ «« · · »« * * · · · »Φ ·· *» φ φ · φ. At least one segment can bind to form double helices of the polypeptide material of claims P1 and P2, and wherein the functional domain can be added to the polypeptide material during the initial composition or later.
[4]
Polypeptide material according to any of claims 1 to 3, characterized in that the elastin-like segment is composed of at least 4 repeats similar to those we have in animal and human elastin, their mutant or synthetic elastin with retained function , similar to elastin.
[5]
Polypeptide material according to any of claims 1 to 4, characterized in that the elastin-like segment is made up of at least 4 elastin-like repeats, including but not limited to: pentapeptides Val-Pro-Gly-Val-Gly or Gly- Val-Gly-Val-Pro or Gly-Val-Gly-Ile-Pro.
[6]
Polypeptide material according to any of claims 1 to 5, characterized in that the elastin-like segment is made up of at least 12 and predominantly less than 600 amino acid residues, the sum (% (n / n glycine residues) and 2 * {% (n / n) proline residues) is more than 60% (n / n),
[7]
7. polypeptide material according to any of claims 1 to 6, characterized in that the number of elastin-like segments is between 1 and 50, preferably between 2 and 10, and the number of segments to form double helixes between 2 and 50, primarily between 3 and 10, wherein the elastin-like segment is at least in one case between segments to form double helices.
[8]
8. polypeptide material according to any one of claims 1 to 7, characterized in that segments are formed to form double helices of at least two heptads and are capable of homo-oligomerates or hetero-oligomerates with a Oligomerisierungszustand 2-7 to form, with segments for Formation of double helices, parallel or antiparallel, can be selected from natural polypeptides or cultured polypeptides.
[9]
9. Polypeptide material according to any of claims 1 to 7, characterized in that segments for forming double helices have been selected 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.
[10]
10. polypeptide material which is constructed according to any of claims 1 to 9 from at least two components, characterized in that the composite polypeptide material was prepared with integration of at least two polypeptide materials according to any of claims 1 to 9, and wherein at least one segment to form double helices from a component of the polypeptide material having at least one of the double helix segments, forms hetero-oligomers, other components of the polypeptide material, which provides a mechanism for controlling the assembly of the composite polypeptide material.
[11]
Polypeptide material according to any of claims 1 to 10, characterized in that it contains at least one elastin-like segment and at least two segments to form double helices which are optionally bound to the functional protein domain, but segments and domains are optional with a linker which binds from one to 20 amino acids, preferably from one to six amino acids, but optionally also contains a signal sequence that directs the secretion of the protein and the amino acid sequence that serves to label.
[12]
The polypeptide material according to claim 3, characterized in that it contains double helix segments united with functional polypeptide domains, double helix segments, and the polypeptide functional domains are optionally linked together with a link containing at least one and up to 20 amino acids , primarily one to 6 amino acids. The protein optionally also contains a signal sequence that controls the secretion of the protein and the amino acid tagging. *** "
[13]
13. polypeptide material which is composed of polypeptide material in different proportions according to any claim from 1 to 12 constructed.
[14]
The decomposition process of polypeptide material according to any of claims 10 to 13, with addition of polypeptide containing a helix capable of regenerating with segments to form double helices in the component of the polypeptide material, and the peptides are preferentially selected from SEQ ID NO: 10 , 12, 28, 30, 32, 34, 36, 38.
[15]
A DNA carrying a record for the polypeptide material of any of claims 1 to 13, wherein the DNA is operably linked to regulatory elements, the promoter and the terminator, with the intention of enhancing expression of polypeptide material in host organisms.
[16]
A preparation process of polypeptide material according to any of claims 1 to 13 which includes the steps of: a) cultivating a host organism expressing polypeptide material as claimed in any of claims 1 to 13 and encoded in the DNA of claim 15; b) isolation of polypeptide material; and c) mixing polypeptide material for the purpose of forming polypeptide material according to any one of claims 1 to 13.
[17]
17. Use of polypeptide material according to any of claims 1 to 13 for medical and pharmaceutical material intended to accelerate cell, tissue and organ growth in vitro.
[18]
18. Use of polypeptide material according to any of claims 1 to 13 for medical and pharmaceutical material intended for the treatment of living tissue, especially human or animal tissue, with applications such as, but not limited to, substitution or regeneration of damaged tissue, such as Prosthesis, dressings for the treatment of wounds and burns, local delivery of cytotoxic or cytostatic polypeptides.
[19]
19. Use of polypeptide material according to any of claims 1 to 13 for medical and pharmaceutical material, intended for growth inhibition of pathogens, especially bacteria, viruses and yeasts. Linz, April 12, 2012 Kemijski

stitut through: C% λΑ ^ ίύί / ΛΑ , d

类似技术:

---

公开号 | 公开日 | 专利标题

---

Loo et al.2015|Self‐assembled proteins and peptides as scaffolds for tissue regeneration

---

Miranda-Nieves et al.2017|Collagen and elastin biomaterials for the fabrication of engineered living tissues

---

Rubert Pérez et al.2015|The powerful functions of peptide-based bioactive matrices for regenerative medicine

---

Maude et al.2013|Biomimetic self-assembling peptides as scaffolds for soft tissue engineering

---

Webber et al.2013|Supramolecular nanofibers of peptide amphiphiles for medicine

---

CA2719250C|2020-04-07|Polypeptide-polymer conjugates and methods of use thereof

---

US20160194378A1|2016-07-07|Peptides and uses thereof

---

Abascal et al.2018|The past, present and future of protein-based materials

---

DE69333040T2|2004-05-06|MORPHOGEN INDUCED NERVES RESTORATION AND RECONSTRUCTION.

---

Janani et al.2019|Insight into silk-based biomaterials: from physicochemical attributes to recent biomedical applications

---

Saric et al.2019|Engineering of silk proteins for materials applications

---

CN101979106B|2013-07-31|Biological material for rehabilitating spinal cord injury and preparation method and application thereof

---

DE69628576T2|2004-04-22|procollagens

---

Huber et al.2014|Introducing a combinatorial DNA-toolbox platform constituting defined protein-based biohybrid-materials

---

EP1151095B1|2010-06-09|Polypeptide variants with raised heparin-binding capacity

---

WO2015168321A9|2016-01-28|Ocular compositions and methods thereof

---

Corchero et al.2014|Recombinant protein materials for bioengineering and nanomedicine

---

WO2017146819A1|2017-08-31|Implantable drug delivery systems

---

Chouhan et al.2019|Silkworm silk matrices coated with functionalized spider silk accelerate healing of diabetic wounds

---

AT506150B1|2010-01-15|CYCLIC AND CYSTONE FREE PEPTIDE

---

Debabov et al.2020|Recombinant spidroins as the basis for new materials

---

AT511187A2|2012-09-15|Polypeptide material from elastin-like segments and coiled-coil segments

---

DE69735533T2|2006-10-12|Soluble polypeptides consisting of the first coiled-coil domain of human and mouse epimorphin

---

US20140031526A1|2014-01-30|High Molecular Ordered Fibrilar Structures, Method for Their Preparation and Uses Thereof

---

DE69837265T2|2007-11-15|POLYPEPTIDES FOR REGENERATING THE NERVOUS SYSTEM

同族专利:

---

公开号 | 公开日

---

WO2011046519A1|2011-04-21|

---

AT511187A5|2015-02-15|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

US20030219451A1|2001-10-29|2003-11-27|Whitehead Institute For Biomedical Research|Stable helical C peptides and uses therefor|

---

US9554997B2|2007-06-18|2017-01-31|New York University|Polymer carrier|CN102360497B|2011-10-19|2013-06-12|西安电子科技大学|SARimage segmentation method based on parallel immune clone clustering|

---

US9332010B2|2014-03-07|2016-05-03|Motorola Solutions, Inc.|Methods and systems for token-based application management|

法律状态:

优先权:

---

申请号 | 申请日 | 专利标题

---

PCT/SI2009/000047|WO2011046519A1|2009-10-12|2009-10-12|Polypeptide material composed of elastin-like segments and coiled coil segments|

---