SI23184A - Polypeptide material composed of elastin-like segments and segments for formation of coiled helices - Google Patents
Polypeptide material composed of elastin-like segments and segments for formation of coiled helices Download PDFInfo
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Polipeptidni material sestavljen iz elastinu podobnih segmentov in segmentov za tvorbo ovitih vijačnicPolypeptide material composed of elastin-like segments and segments for forming wrapped helices
Področje izumaFIELD OF THE INVENTION
Področje izuma je nov polipeptiden material, sestavljen iz elastinu podobnih segmentov, segmentov za tvorbo ovitih vijačnic in izbirno tudi funkcionalnih polipeptidnih domen, ki opredelijo možne uporabe polipeptidnega materiala. Področje izuma je protein, ki tvori polipeptidni material in DNK, ki zapisuje protein, ki tvori polipeptidni material. Področje izuma je tudi uporaba polipeptidnega materiala za spodbujanje rasti celic, tkiv ali organov, za zaviranje rasti patogenov in za medicinski oziroma farmacevtski material uporaben za zdravljenje živega tkiva, prednostno človeškega ali živalskega tkiva.The field of the invention is a novel polypeptide material consisting of elastin-like segments, segments for forming wrapped helices and optionally also functional polypeptide domains, which define the possible uses of the polypeptide material. The field of the invention is a protein that forms a polypeptide material and DNA that writes a protein that forms a polypeptide material. The field of the invention is also the use of a polypeptide material for promoting the growth of cells, tissues or organs, for inhibiting the growth of pathogens and for medical or pharmaceutical material useful for the treatment of living tissue, preferably human or animal tissue.
Stanje tehnikeState of the art
Za biokompatibilno podlago za rast celic in tkiv so bili razviti različni materiali, ki so pomembni ne le za gojenje celic in vitro, ampak tudi za popravljanje tkiva in ostale aplikacije tkivnega inženirstva ter za aplikacije v medicini. Kot podlaga za delitev, migracijo in diferenciacijo celic lahko služijo številni sintetični polimeri (na primer polistireni, polietilen vinil acetati, polipropileni, polietileni, ipd.) in biopolimeri (vključno s proteini, kot sta kolagen in fibrin, ter polisaharidi, kot so glukozaminoglikani in alginat).For a 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 repair and other tissue engineering applications and for medical applications. A number of synthetic polymers (eg polystyrenes, polyethylene vinyl acetates, polypropylenes, polyethylenes, etc.) and biopolymers (including proteins such as collagen and fibrin, and polysaccharides such as glucosaminoglycans and can serve as a basis for cell division, migration and differentiation) alginate).
Racionalno načrtovani peptidi ponujajo obetaven pristop h konstruiranju biomaterialov. Poročali so na primer že o več primerih novih samosestavljivih in samih sebi komplementarnih amfifilnih peptidov z značilnim izmenjevanjem hidrofobnih in hidrofilnih aminokislin. Ti peptidi tvorijo različne strukture, ki temeljijo na nastajanju fibril s strukturo β-lista med amfifili (U.S. Pat. Appl. Pub. 0209145 Al) in so lahko združeni tudi z biološko aktivnimi komponentami in/ali hidrofobnimi alkilnimi repi na terminalnih delih (U.S. Pat. Appl. Pub. 0181973 Al, U.S. Pat. 7371719 B2).Rationally designed peptides offer a promising approach to the construction of biomaterials. For example, several cases of new self-assembled and self-complementary amphiphilic peptides with characteristic exchange of hydrophobic and hydrophilic amino acids have been reported. These peptides form different structures based on the formation of fibrils with a β-leaf structure between amphiphiles (US Pat. Appl. Pub. 0209145 A1) and can also be combined with biologically active components and / or hydrophobic alkyl tails on the terminal parts (US Pat. Appl. Pub. 0181973 Al, US Pat. No. 7371719 B2).
Biokompatibilni materiali, ki posnemajo ekstracelulami matriks (ECM), predstavljajo ugodno okolje za rast celic. Ekstracelulami matriks (ECM) sestavljajo heterogene makromolekule vključno s proteini in polisaharidi, ki tvorijo tridimenzionalno okolje za rast celic, kar predstavlja podlago za stabilizacijo in podporo celičnih slojev in tkiv.Biocompatible materials that mimic the extracellular matrix (ECM) provide a favorable environment for cell growth. Extracellular matrix (ECM) consists 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 tissues.
Ena od sestavin ECM so elastična vlakna, sestavljena iz amorfnega proteina elastina in mikrofibrile sestavljene večinoma iz fibrilina-1. Prekurzor stabilnega, zamreženega elastina je topna molekula tropoelastin. Ta molekula je sestavljena iz dveh tipov domen: hidrofobnih domen z veliko Gly (G), Val (V), Ala (A) in Pro (P) aminokislinskih ostankov, ki se pogosto pojavljajo v ponavljajočih se zaporedjih (prva določena so bila pentapeptid VPGVG, heksapeptid VGVAPG in tetrapeptid VPGG) ter hidrofilnih domen, ki vsebujejo predvsem Ala in Lys ostanke, pomembne za prečno povezovanje, ki vodi do nastanka netopnega in zelo stabilnega polimera.One of the components of ECM are elastic fibers composed of the amorphous protein elastin and microfibrils composed mainly of fibrillin-1. The precursor of stable, cross-linked elastin is the soluble molecule tropoelastin. This molecule consists of two types of domains: hydrophobic domains with many Gly (G), Val (V), Ala (A) and Pro (P) amino acid residues, which often occur in repetitive sequences. The first to be identified was the pentapeptide VPGVG , the hexapeptide VGVAPG and the tetrapeptide VPGG) and hydrophilic domains containing mainly Ala and Lys residues important for cross - linking, leading to the formation of an insoluble and highly stable polymer.
Tropoelastin in polimeri sorodni elastinu lahko tvorijo koacervate preko hidrofobnih domen preden se molekule kovalentno prečno povežejo (proces, ki ga sproži povišanje temperature), kar nadalje vodi do samosestavljanja. Nagnjenost k samoagregaciji in pomembna stabilnost elastina in elastinu sorodnih polimerov določata elastin kot odlično komponento za razvoj sintetičnih nanomaterialov.Tropoelastin and elastin-related polymers can form coacervates via hydrophobic domains before the molecules covalently cross-link (a process triggered by a rise in temperature), further leading to self-assembly. The tendency to self-aggregation and the significant stability of elastin and elastin-related polymers define elastin as an excellent component for the development of synthetic nanomaterials.
Lee in sodelavci (Lee et al., 2001, Biomacromolecules. 2, 170-179) so podrobno analizirali polipeptide z elastinu podobnimi hidrofobnimi ponovljivimi zaporedji, ki lahko služijo kot topni modeli za elastin. Za uporabo teh polipeptidov kot odpornih biomaterialov, pa je nujna prečna povezava polipeptidnih verig. To je možno doseči z uporabo γ-žarkov, kemičnim povezovanjem, lahko pa je povezovanje tudi encimsko posredovano. Pripravljenih je bilo že nekaj elastinu podobnih polipeptidnih materialov, kjer so uvedli nekaj modifikacij v hidrofobne ponavljajoče sekvence, vključno z zamenjavo nekaterih aminokislin z ostanki, ki bi lahko služili za prečno povezovanje, ali dodajanjem komponent za prečno povezovanje (substrat za lizil oksidazo) elastomemi komponenti (tetra oz. pentapeptidne ponavljajoče enote ali njihove mešanice) (U.S. Pat. 4589882). Da bi dobili sistem z boljšim prečnim povezovanjem, so pripravili tudi material, ki ima proste amino skupine na enem intermediatu VPGVG peptida in proste karboksilne skupine na drugem , kar omogoča prečno povezovanje s kemičnim reagentom (U.S. Pat. 4187852). Z uporabo rekombinantne tehnologije so pripravili multimodularne polipeptide, ki temeljijo na elastinu in lahko spreminjajo mehanske in funkcionalne lastnosti materialov, ki temeljijo na elastinu. Primeri vključujejo hibride med elastinu podobnimi peptidi in C5 domeno -3- fibronektina, ki pospešuje pritrjanje celic (Welsh et al., 2000, Biomacromolecules. 1, 23-30), ali hibride med peptidi podobnimi fibroinu iz svile in elastinu podobnimi peptidi (Cappello et al., 1990, Biotechnology Progress. 6, 198-202). Elastin in elastinu podobne komponente veliko obetajo na področju nano-biomaterialov (lahko predstavljajo substrat za rast celic ali material za dostavo zdravil in rastnih faktorjev) in tkivnega inžinirstva (tudi kot nadomestki za kožo, krvne žile, srčne zaklopke in elastični hrustanec).Lee et al. (Lee et al., 2001, Biomacromolecules. 2, 170-179) analyzed in detail polypeptides with elastin-like hydrophobic repeatable sequences that can serve as soluble models for elastin. However, for the use of these polypeptides as resistant biomaterials, cross-linking of the polypeptide chains is essential. This can be achieved by using γ-rays, chemical binding, but the binding can also be enzymatically mediated. Several elastin-like polypeptide materials have already been prepared, where several modifications to hydrophobic repeating sequences have been introduced, including the replacement of certain amino acids with residues that could serve as cross-linking or the addition of cross-linking components (lysyl oxidase substrate) elastomemi components (tetra or pentapeptide repeating units or mixtures thereof) (US Pat. 4589882). To obtain a system with better cross-linking, a material having free amino groups on one intermediate of the VPGVG peptide and free carboxyl groups on the other was also prepared, allowing cross-linking with a chemical reagent (U.S. Pat. 4187852). Using recombinant technology, elastin-based multimodular polypeptides have been developed that can alter the mechanical and functional properties of elastin-based materials. Examples include hybrids between elastin-like peptides and the C5 domain of -3-fibronectin, which promotes cell attachment (Welsh et al., 2000, Biomacromolecules. 1, 23-30), or hybrids between silk fibroin-like peptides and elastin-like peptides (Cappello et al., 1990, Biotechnology Progress. 6, 198-202). Elastin and elastin-like components hold great promise in the field of nano-biomaterials (they can be a substrate for cell growth or a material for drug delivery and growth factors) and tissue engineering (also as substitutes for skin, blood vessels, heart valves, and elastic cartilage).
Sestavljen material lahko vsebuje tudi različne dodatne funkcionalne biološko aktivne dele, zaradi česar lahko vpliva na pritrjanje, adhezijo, migracijo ali proliferacijo celic. Možnost pritrjanja funkcionalnih biološko aktivnih delov na ogrodje predstavlja pomembno lastnost pri konstruiranju samosestavljivih peptidov. Ta način nam omogoča ciljanje tarčne celice in zmanjša količino substance, ki je potrebna, da dosežemo zaželen lokalni efekt. Dostava nekaterih rastnih faktorjev v topni obliki lahko predstavlja dodatno slabost, saj celice niso več odzivne na faktor zaradi intemalizacije le-tega in zaradi regulacije v obliki zmanjšanega števila receptorjev za rastni faktor.The composite material may also contain various additional functional biologically active parts, which may affect the attachment, adhesion, migration or proliferation of cells. The possibility of attaching functional biologically active parts to the framework is an important property in the construction of self-assembled peptides. This method allows us to target the target cell and reduces the amount of substance needed to achieve the desired local effect. The delivery of some growth factors in soluble form may present an additional disadvantage, as cells are no longer responsive to the factor due to its actualization and due to regulation in the form of a reduced number of growth factor receptors.
Izum se nanaša na polipeptidni material sestavljen iz segmentov podobnih elastinu, ki so med seboj prečno povezani preko nekovalentnih interakcij z uporabo novega pristopa izbranih oziroma načrtovanih segmentov za tvorbo ovitih vijačnic, s čimer se izognemo potrebi po kemijski spremembi elastinu podobnih ponovitev z uvedbo mest za prečno povezovanje. Izum je nadalje izpopolnjen z uporabo reguliranega sestavljanja in razstavljanja polipeptidnega materiala. Polipeptidni material se lahko tudi dodatno izpopolni z dodanimi funkcionalnimi proteinskimi domenami za pospeševanje celične rasti, diferenciacijo, inhibicijo mikrobne rasti, vezavo kovinskih ionov, citotoksičnost, reprogramiranje celic ali mnogo drugih funkcij in zato predstavlja nov biomaterial za rast celic/tkiv/organov in za zdravljenje živega človeškega ali. živalskega tkiva. Tak pristop omogoča skoraj nešteto možnih kombinacij, poleg tega pa omogoča tudi pritrjanje funkcionalnih proteinskih domen na že sestavljen biomaterial po začetnem sestavljaju, kar lahko omogoča načrtovanje časovnega poteka terapije.The invention relates to a polypeptide material composed of elastin-like segments that are transversely interconnected through non-covalent interactions using a new approach of selected or designed segments to form wrapped helices, thus avoiding the need for chemical modification of elastin-like repeats by introducing transverse sites. connecting. The invention is further improved by the use of controlled assembly and disassembly of the polypeptide material. The polypeptide material can also be further enhanced with added functional protein domains to promote cell growth, differentiation, microbial growth inhibition, metal ion binding, cytotoxicity, cell reprogramming or many other functions and therefore represents a new biomaterial for cell / tissue / organ growth and treatment living human or. animal tissue. Such an approach allows for almost a myriad of possible combinations, and it also allows the attachment of functional protein domains to an already assembled biomaterial after initial assembly, which can allow for planning the timing of therapy.
Povzetek izumaSummary of the invention
Izum se nanaša na polipeptidni material, ki vsebuje vsaj en elastinu podoben segment in vsaj dva segmenta za tvorbo ovitih vijačnic, pri čemer se vsaj en elastinu podoben segment nahaja med dvema ali več segmenti za tvorbo ovitih vijačnic, ločene polipeptidne molekule pa se med sabo povezujejo preko interakcij med segmenti za tvorbo ovitih vijačnic in se nahajajo na teh molekulah. Izum se nanaša tudi na polipeptidni material, ki izbirno vsebuje vsaj eno funkcionalno polipeptidno domeno, izbrano med, vendar ne omejeno na: rastne faktorje, faktorje za celično diferenciacijo, protimikrobne peptide, domene za vezavo kovin in inhibitorje bakterijske rasti. Rastni faktorji so izbrani prednostno, vendar ne omejeno izmed: epidermalnih rastnih faktorjev (EGF), fibroblastnih rastnih faktorjev in rastnih faktorjev, ki spodbujajo rast nevronov, prednostno nevronski rastni factor (NGF). Protimikrobni peptidi so izbrani prednostno, vendar ne omejeno izmed, katelicidinov in defenzinov, prednostno katelicidin LL-37. Domene za vezavo kovin so na primer AEA ali heksa histidinski rep, prednostno SEQ ID NO: 26. Ena ali več funkcionalnih domen je lahko kovalentno vezanih na polipeptidni material ali pa so v polipeptidni material uvedene preko interakcij med segmenti za tvorbo ovitih vijačnic, ki so spojeni s funkcionalno domeno, in segmenti za tvorbo ovitih vijačnic, ki so del polipeptidnega materiala.The invention relates to a polypeptide material comprising at least one elastin-like segment and at least two segments for forming coiled helices, wherein at least one elastin-like segment is located between two or more segments for forming coiled helices, and the separate polypeptide molecules are interconnected through interactions between segments to form coiled helices and located on these molecules. The invention also relates to a polypeptide material optionally comprising at least one functional polypeptide domain selected from, but not limited to: growth factors, cell differentiation factors, antimicrobial peptides, metal binding domains, and bacterial growth inhibitors. Growth factors are selected preferably, but not limited to: epidermal growth factors (EGF), fibroblast growth factors, and neuronal growth-promoting factors, preferably neuronal growth factor (NGF). Antimicrobial peptides are preferably selected, but not limited to, cathelicidins and defensins, preferably cathelicidin LL-37. The metal binding domains are, for example, AEA or hexa histidine tail, preferably SEQ ID NO: 26. One or more functional domains may be covalently bound to the polypeptide material or introduced into the polypeptide material through interactions between the helix-forming helix segments that are fused to a functional domain, and segments to form coiled helices that are part of the polypeptide material.
Izum obsega tudi polipeptidni material sestavljen iz različnih razmerij polipeptidnih komponent, ki lahko vključujejo tudi različne funkcionalne domene. V skladu z izumom vsebuje polipeptidni material vsaj en elastinu podoben segment in vsaj dva segmenta za tvorbo ovitih vijačnic in izbirno vsaj eno funkcionalno polipeptidno domeno, pri čemer je elastinu podoben segment človeški ali živalski elastin, njegova mutanta ali sintetičen elastinu podoben segment z ohranjenimi funkcionalnimi lastnostmi elastina in ga sestavljajo vsaj 4 elastinu podobne ponovitve, vključno z, vendar ne omejene na: pentapeptid Val-Pro-Gly-Val-Gly ali Gly-Val-Gly-Val-Pro ali Gly-Val-Gly-Ile-Pro. Dodatno je elastinu podoben segment v tem izumu opredeljen s tem, da ga sestavlja vsaj 12 aminokislinskih ostankov, pri čemer je vsota (% (n/n) glicinskih ostankov) in 2* (% (n/n) prolinskih ostankov) višja od 60 % (n/n).The invention also encompasses a polypeptide material composed of different ratios of polypeptide components, which may also include different functional domains. According to the invention, the polypeptide material comprises at least one elastin-like segment and at least two segments for forming coiled helices 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 with retained functional properties elastin and consists of at least 4 elastin-like repeats, including, but not limited to: the pentapeptide Val-Pro-Gly-Val-Gly or Gly-Val-Gly-Val-Pro or Gly-Val-Gly-Ile-Pro. Additionally, the elastin-like segment in the present invention is defined in that it consists of at least 12 amino acid residues, the sum of (% (n / n) glycine residues) and 2 * (% (n / n) proline residues) being greater than 60 % (n / n).
Izum se nanaša tudi na polipeptidni material opisan zgoraj, kjer je število elastinu podobnih segmentov med 1 in 50, prednostno med 2 in 10, in število segmentov za tvorbo ovitih vijačnic med 2 in 50, prednostno med 3 in 10, in kjer je vsaj v enem primeru elastinu podoben segment umeščen med segmenta za tvorbo ovitih vijačnic.The invention also relates to the polypeptide material described above, wherein the number of elastin-like segments is between 1 and 50, preferably between 2 and 10, and the number of segments for forming wrapped helices is between 2 and 50, preferably between 3 and 10, and where at least in in one case, an elastin-like segment is placed between the segments to form the helixes.
Iznum se nanaša na polipeptidni material opisan zgoraj, kjer segmente za tvorbo ovitih vijačnic predstavljajo naravni ali načrtovani motivi za tvorbo ovitih vijačnic, in so sestavljeni iz vsaj dveh heptad in so lahko paralelni ali antiparalelni in tvorijo ali homooligomere z oligomerizacijskim stanjem med 2 in 7 ali heterooligomere z oligomerizacijskim stanjem med 2 in 7.The invention relates to a polypeptide material described above, wherein the enveloped helix formation segments represent natural or intended motifs for enveloped helix formation, and consist of at least two heptads and may be parallel or antiparallel and form either oligomerization states with an oligomerization state between 2 and 7 or heterooligomers with an oligomerization state between 2 and 7.
Izum se nanaša na polipeptidni material, kjer so segmenti za tvorbo ovitih vijačnic, ki povezujejo različne molekule, izbrani izmed načrtovanih parov peptidov za tvorbo ovitih vijačnic: SEQ ID: 10 in SEQ ID: 12, SEQ ID: 28 in SEQ ID: 30; SEQ ID:32 in SEQ ID: 34, SEQ ID: 36 in SEQ ID: 38 ali SEQ ID: 14 ali SEQ ID: 26.The invention relates to a polypeptide material, wherein the wrapped helix-forming segments connecting the various molecules are selected from the intended pairs of wrapped helix-forming peptides: 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.
Izum se nanaša na polipeptidni material sestavljen iz vsaj dveh različnih polipeptidnih materialov opisanih zgoraj, pri čemer je sestavljen polipeptidni material pripravljen z združevanjem vsaj dveh polipeptidnih materialov in pri čemer segmenti za tvorbo ovitih vijačnic iz enega materiala lahko tvorijo heterooligomere s segmenti za tvorbo ovitih vijačnic iz drugega materiala, s čimer dosežemo regulirano povezovanje sestavljenega polipeptidnega materiala.The invention relates to a polypeptide material composed of at least two different polypeptide materials described above, wherein the composite polypeptide material is prepared by combining at least two polypeptide materials, and wherein the helix-forming helix segments can form heterooligomers with helix-forming helix segments. of another material, thereby achieving regulated binding of the composite polypeptide material.
Izum se nanaša na peptide, ki vsebujejo segmente za tvorbo ovitih vijačnic in so prisotni samo v enem od komponent polipeptidnega materiala opisanega zgoraj in jih lahko uporabimo za razstavljanje polipeptidnega materiala, kar omogoča nežen postopek za ločevanje celic od materiala.The invention relates to peptides containing segments for forming helixes and present in only one of the components of the polypeptide material described above and can be used to disassemble the polypeptide material, allowing a gentle process for separating cells from the material.
Izum se nanaša na polipeptidni material opisan zgoraj, pri čemer so segmenti in domene v zgoraj omenjenem polipeptidnem materialu izbirno povezani med seboj s povezovalnim delom, ki vsebuje od ene do 20 aminokislin, prednostno od ene do 6 aminokislin, in pri čemer protein izbirno vsebuje signalno zaporedje, ki usmerja izločanje proteina, in aminokislinski/e označevalec/ce.The invention relates to the polypeptide material described above, wherein the segments and domains in the above-mentioned polypeptide material are optionally linked to each other by a binding moiety containing from one to 20 amino acids, preferably from one to 6 amino acids, and wherein the protein optionally contains a signaling the sequence that directs protein secretion and the amino acid marker (s).
Izum se nanaša na DNK, ki nosi zapis za proteine opisane zgoraj, pri čemer je DNK operativno povezana z regulatiomimi elementi, promotorjem in terminatorjem, ki omogočajo izražanje fuzijskega proteina v gostiteljskem organizmu.The invention relates to DNA carrying the transcript for the proteins described above, wherein the DNA is operatively linked to regulatory elements, a promoter and a terminator that allow the expression of a fusion protein in a host organism.
Izum se nanaša na proces priprave zgoraj opisanega polipeptidnega materiala, ki vključuje naslednje korake: a) gojenje gostiteljskega organizma, ki izraža protein, ki ga zapisuje DNK, oboje opisano zgoraj; b) izolacijo izraženega proteina; in c) tvorbo polipeptidnega materiala opisanega zgoraj z mešanjem očiščenega/ih proteina/ov.The invention relates to a process for the preparation of the polypeptide material described above, comprising the following steps: a) culturing a host organism expressing a protein recorded by DNA, both described above; b) isolating the expressed protein; and c) forming the polypeptide material described above by mixing the purified protein (s).
Izum se nanaša na uporabo zgoraj opisanega polipeptidnega materiala za rast celic, tkiv ali organov, pri čemer ta material izbirno zagotavlja zaželene funkcionalne lastnosti za rast celic. Izum se nanaša tudi na uporabo zgoraj opisanega polipeptidnega materiala kot medicinski in -6- farmacevtski material za zdravljenje živega tkiva, prednostno človeškega ali živalskega tkiva. Na primer, vendar ne omejeno na nadomeščanje poškodovanega tkiva, kot proteza, za regeneracijo celic, reprogamiranje celic in kot farmacevtski material, kot npr. ovoji za zdravljenje ran in opeklin, lokalno dostavo citotoksičnih ali citostatičnih polipeptidov.The invention relates to the use of the polypeptide material described above for the growth of cells, tissues or organs, this material optionally providing the desired functional properties for cell growth. The invention also relates to the use of the polypeptide material described above as a medical and -6- pharmaceutical material for the treatment of living tissue, preferably human or animal tissue. For example, but not limited to replacing damaged tissue, as a prosthesis, for cell regeneration, cell reprogramming, and as a pharmaceutical material, e.g. dressings for the treatment of wounds and burns, local delivery of cytotoxic or cytostatic polypeptides.
Izum se nanaša na uporabo zgoraj opisanega polipeptidnega materiala za inhibicijo rasti patogenov v primeru da so funkcionalne polipeptidne domene polipeptidnega materiala protimikrobni peptidi ali domene za vezavo kovin, še posebno SEQ ID NO: 26., ki tvori srebrne nanodelce.The invention relates to the use of the polypeptide material described above for inhibiting the growth of pathogens in the case where the functional polypeptide domains of the polypeptide material are antimicrobial peptides or metal binding domains, in particular SEQ ID NO: 26., which forms silver nanoparticles.
Kazalo slikIndex images
Slika 1: Shematska predstavitev iznajdbe. Polipeptidni material je sestavljen iz elastinu podobnih segmentov in segmentov, ki tvorijo ovite vijačnice. Odseki, ki tvorijo ovite vijačnice, oligomerizirajo in povežejo posamezne polipeptidne verige polipeptidnega ogrodja biomateriala. Funkcionalne polipeptidne domene so lahko vključene v polipeptidno verigo kot del fuzijskega proteina, ali pa so povezane z ogrodjem preko interakcij med segmenti, ki tvorijo ovite vijačnice na ogrodju in tistimi, ki so pripojeni funkcionalnemu polipeptidu.Figure 1: Schematic representation of the invention. The polypeptide material consists of elastin-like segments and segments that form coiled helices. The sections forming the wrapped helices oligomerize and link the individual polypeptide chains of the polypeptide backbone of the biomaterial. Functional polypeptide domains may be incorporated into the polypeptide chain as part of a fusion protein, or they may be linked to the framework through interactions between the segments that form the enveloped helices on the framework and those attached to the functional polypeptide.
Slika 2: Protimikrobno delovanje polipeptidnega materiala, ki vsebuje funkcionalno polipeptidno domeno - protimikrobni peptid LL-37. Na sliki so sledeči vzorci: l-MilliQ, 2-6 naraščajoče koncentracije polipeptidnega materiala, ki vsebuje funkcionalno polipeptidno domeno - protimikrobni peptid 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 2: Antimicrobial activity of a polypeptide material containing a functional polypeptide domain - antimicrobial peptide LL-37. The figure shows the following samples: l-MilliQ, 2-6 increasing concentrations of polypeptide material containing a functional polypeptide domain - antimicrobial peptide LL-37; 2- 0.1 mg / ml, 3- 0.5 mg / ml, 4- 1 mg / ml, 5- 5 mg / ml, 6-10 mg / ml.
Slika 3: Diferenciacija PC 12 celic na polipeptidnem materialu, ki vsebuje funkcionalno polipeptidno domeno - rastni faktor NGF. Na sliki so prikazane: [A, Β] HEK293T celice na polipeptidnem materialu [Α] brez funkcionalne polipeptidne domene, [Β] s funkcionalno polipeptidno domeno - rastnim faktorjem NGF; [C, D] PC12 celice na polipeptidnem materialu [C] brez funkcionalne polipeptidne domene, [D] s funkcionalno polipeptidno domeno - rastnim faktorjem NGF .Figure 3: Differentiation of PC 12 cells on polypeptide material containing a functional polypeptide domain - NGF growth factor. The figure shows: [A, [] HEK293T cells on polypeptide material [Α] without functional polypeptide domain, [Β] with functional polypeptide domain - growth factor NGF; [C, D] PC12 cells on polypeptide material [C] without functional polypeptide domain, [D] with functional polypeptide domain - NGF growth factor.
Slika 4: Protimikrobno delovanje polipeptidnega materiala, ki vsebuje funkcionalno polipeptidno domeno AEA, ki tvori srebrne nanodelce. Slika kaže, da funkcionalna polipeptidna domena, ki tvori srebrne nanodelce, zavira bakterijsko rast. -7-Figure 4: Antimicrobial activity of a polypeptide material containing a functional polypeptide domain of AEA forming silver nanoparticles. The figure shows that the functional polypeptide domain that forms silver nanoparticles inhibits bacterial growth. -7-
Slika 5: CD spekter dokazuje tvorbo heterodimerov reprezentativnega para načrtovanih segmetov, ki tvorita ovito vijačnico, Pl in P2.Figure 5: The CD spectrum demonstrates the formation of heterodimers of a representative pair of designed segments forming an enveloped helix, Pl and P2.
Podroben opis izuma Če ni definirano drugače, imajo vsi tehnični in znanstveni izrazi uporabljeni tukaj enak pomen kot je splošno sprejet med strokovnjaki na področju izuma. Terminologija, ki je uporabljena v opisu izuma, je namenjena opisovanju določenega dela izuma in ne omejevanju izuma. Vse objave omenjene v opisu so navedene med referencami. V opisu izuma in v zahtevkih je opis v ednini, vendar opis vključuje tudi množino, ki pa ni poudarjena zaradi lažjega razumevanja.DETAILED DESCRIPTION OF THE INVENTION Unless defined otherwise, all technical and scientific terms used herein have the same meaning as generally accepted among those skilled in the art. The terminology used in the description of the invention is intended to describe a particular part of the invention and not to limit the invention. All publications mentioned in the description are listed among the references. In the description of the invention and in the claims, the description is in the singular, but the description also includes the plural, which is not emphasized for ease of understanding.
Polipeptidni materialPolypeptide material
Osnova izuma je odkritje, da lahko vsaj en elastinu podoben segment z vsaj dvema segmentoma za tvorbo ovitih vijačnic tvori polipeptidni material. Predstavljen izum opisuje polipeptidni material, katerega sestava temelji na nedavnih dognanjih izumiteljev na področju nano-materialov. Izum temelji na odkritju, da protein sestavljen iz vsaj enega elastinu podobnega segmenta in vsaj dveh segmentov za tvorbo ovitih vijačnic sestavlja polipeptidni material s specifičnimi fizičnimi lastnostmi, npr. spodbujanje rasti celic, tkiv ali organov. Segmenti podobni elastinu posnemajo elastin, zagotavljajo elastomeme lastnosti in so povezani preko oligomerizacije segmentov za tvorbo ovitih vijačnic, kar vodi do tvorbe polipeptidnega materiala. Polipeptidni material zato posnema ekstracelulami matriks, je biokompatibilen, usmerja rast celic in zagotavlja ustrezno okolje za delovanje celic.The basis of the invention is the discovery that at least one elastin-like segment with at least two segments for forming wrapped helices can form a polypeptide material. The present invention describes a polypeptide material whose composition is based on the recent findings of the inventors in the field of nano-materials. The invention is based on the discovery that a protein composed of at least one elastin-like segment and at least two segments for the formation of coiled helices consists of a polypeptide material with specific physical properties, e.g. promoting the growth of cells, tissues or organs. Elastin-like segments mimic elastin, provide elastomeric properties, and are linked through oligomerization of segments to form coiled helices, leading to the formation of polypeptide material. The polypeptide material therefore mimics the extracellular matrix, is biocompatible, directs cell growth, and provides an appropriate environment for cell function.
Izraz "polipeptidni material" v opisu izuma se nanaša na material sestavljen iz proteinov, ki vsebujejo vsaj en elastinu podoben segment in vsaj dva segmenta za tvorbo ovitih vijačnic in čigar struktura je dvo- ali tri-dimenzionalna. Segmenti podobni elastinu materialu zagotavljajo elastomeme lastnosti, medtem ko segmenti za tvorbo ovitih vijačnic omogočajo medsebojno povezovanje elastinu podobnih segmentov.The term " polypeptide material " in the description of the invention it refers to a material composed of proteins comprising at least one elastin-like segment and at least two segments for forming wrapped helices and whose structure is two- or three-dimensional. Elastin-like segments provide elastomeric properties to the material, while segments for forming wrapped helices allow elastin-like segments to interconnect.
Izumitelji so prišli do spoznanja, da je mogoče elastinu podobne ponovitve povezati v polipeptidni material z novim pristopom uporabe segmentov za tvorbo ovitih vijačnic, ki oligomerizirajo. To predstavlja izboljšavo prejšnjih odkritij, saj je bilo prej potrebno za -8- zamreženje elastinu podobne ponovitve modificirati z uvedbo možnih mest za prečno povezovanje (U.S. Pat. 4589882; U.S. Pat. 4187852).The inventors have come to the realization that elastin-like repeats can be linked into a polypeptide material with a new approach to using segments to form oligomerizing coils. This represents an improvement on previous discoveries, as previously the -8- crosslinking of elastin-like repeats had to be modified by introducing possible cross-linking sites (U.S. Pat. 4589882; U.S. Pat. 4187852).
Izraz "elastinu podoben segment" v opisu izuma ima splošen pomen in se nanaša na homologne odseke živalskega ali človeškega elastina sestavljene iz vsaj 4 elastinu podobnih ponovitev. Elastinu podobni segmenti so lahko nanizani eden za drugim ali so lahko vsaj v enem primeru nameščeni med dva segmenta za tvorbo ovitih vijačnic. Izraz "elastinu podoben segment" se nanaša tudi na mutiran ali sintetičen, umetno narejen, elastinu podoben segment, ki ohranja karakteristike elastina. Elastinu podobne segmente je mogoče kemično sintetizirati ali rekombinamtno izraziti. Izraz "homologni odseki" v opisu se nanaša na aminokislinsko zaporedje proteina, ki izvira iz istega ali drugega organizma in kaže veliko podobnost ob poravnavi proteina, prednostno več kot 50 % ohranjene strukture, prednostno 60 %, prednostno 70 % v poravnavi zaporedja. Izraz "homologni odseki" se nanaša tudi na mutirane proteinske odseke, katerih mutacije minimalno spremenijo aminokislinsko zaporedje.The term " elastin-like segment " in the description of the invention has a general meaning and refers to homologous sections of animal or human elastin composed of at least 4 elastin-like repeats. Elastin-like segments may be stacked one after the other or may be placed between two segments to form wrapped helices in at least one case. The term " elastin-like segment " it also refers to a mutated or synthetic, artificially made, elastin-like segment that retains the characteristics of elastin. Elastin-like segments can be chemically synthesized or recombinantly expressed. The term " homologous segments " in the description it refers to the amino acid sequence of a protein originating from the same or another organism and showing great similarity when aligning the protein, preferably more than 50% of the preserved structure, preferably 60%, preferably 70% in sequence alignment. The term " homologous segments " it also refers to mutated protein segments whose mutations minimally alter the amino acid sequence.
Izraz "elastinu podobne ponovitve" se nanaša tudi na hidrofobna ponovljiva zaporedja prisotna v elastinu, ki vsebujejo veliko Gly (G), Val (V), Ala (A) in Pro (P) ostankov, ki se pogosto pojavljajo v tandemskih ponovitvah nekaj (od 3 do 6) aminokislin. Izraz "elastinu podobne ponovitve" vključuje, vendar ni omejen na, pentapeptide z zaporedjem Val-Pro-Gly-Val-Gly ali Gly-Val-Gly-Val-Pro ali Gly-Val-Gly-Ile-Pro.The term " elastin-like repeats " also refers to hydrophobic repeatable sequences present in elastin, which contain many Gly (G), Val (V), Ala (A) and Pro (P) residues, which often occur in tandem repeats of some (3 to 6) amino acids . The term " elastin-like repeats " includes, but is not limited to, pentapeptides of the sequence Val-Pro-Gly-Val-Gly or Gly-Val-Gly-Val-Pro or Gly-Val-Gly-Ile-Pro.
Izraz "elastinu podobne ponovitve" se prav tako nanaša na zaporedja sestavljena iz vsaj 12 aminokislinskih ostankov, kjer je vsota (% (n/n) glicinskih ostankov) in 2* (% (n/n) prolinskih ostankov) višja kot 60 % (n/n). Elastomema funkcija elastina je namreč odvisna od samo dveh vrst aminokislinskih ostankov - glicina in prolina. Te lastnosti elastina postanejo opazne nad določeno mejno vsoto prolina in glicina. Druga lastnost elastomernih domen z vsebnostjo glicina in prolina nad omenjeno mejo je odsotnost α-vijačnice in β-lista, polipeptid ostane v neurejeni obliki, tudi kadar agregira (Rauscher et al. 2006, Structure. 14, 1667-1676).The term " elastin-like repeats " also refers to sequences consisting of at least 12 amino acid residues where the sum of (% (n / n) glycine residues) and 2 * (% (n / n) proline residues) is higher than 60% (n / n). The elastomy function of elastin depends on only two types of amino acid residues - glycine and proline. These properties of elastin become noticeable above a certain limit of proline and glycine. Another property of elastomeric domains with glycine and proline content above said limit is the absence of α-helix and β-sheet, the polypeptide remains in disordered form even when aggregated (Rauscher et al. 2006, Structure. 14, 1667-1676).
Temelj izuma je tudi odkritje, da je število elastinu podobnih segmentov med 1 in 50, prednostno med 2 in 10, in število segmentov za tvorbo ovitih vijačnic, med 2 in 50, prednostno med 3 in 10, pri čemer je vsaj v enem primeru elastinu podoben segment umeščen med segmenta za tvorbo ovitih vijačnic, segmenti za tvorbo ovitih vijačnic pa tvorijo homooligomere ali heterooligomere z oligomerizacijskim stanjem med 2 in 7.. -9- • · · ·The invention is also based on 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 forming wrapped helices, between 2 and 50, preferably between 3 and 10, wherein in at least one case the elastin a similar segment is located between the segments for the formation of wrapped helices, and the segments for the formation of wrapped helices form homooligomers or heterooligomers with an oligomerization state between 2 and 7. -9- • · · ·
Izraz "homooligomerizacija" v opisu izuma ima splošen pomen in se nanaša na proces tvorbe kompleksov sestavljenih iz enega samega tipa monomerov, pri čemer je število monomerov med 2 in 7.The term " homooligomerization " in the description of the invention has a general meaning and refers to the process of formation of complexes consisting of a single type of monomers, where the number of monomers is between 2 and 7.
Izraz "heterooligomerizacija" v opisu izuma ima splošen pomen in se nanaša na proces tvorjenja kompleksov sestavljenih iz različnih tipov monomerov, pri čemer je število monomerov med 2 in 7.The term " heterooligomerization " in the description of the invention has a general meaning and relates to the process of forming complexes composed of different types of monomers, the number of monomers being between 2 and 7.
Izraz "monomer" v zgornjem opisu se nanaša na en segment, ki tvori ovito vijačnico in lahko interagira z drugim segmentom, ki tvori ovito vijačnico, tako, da se ovijeta eden okrog drugega.The term " monomer " in the above description refers to one segment forming a wrapped helix and can interact with another segment forming a wrapped helix by wrapping around each other.
Izraz "segment za tvorbo ovitih vijačnic" ima splošen pomen in se nanaša na strukturne proteinske motive, sestavljene iz 2 ali več α-vijačnic, ki se ovijejo ena okrog druge in tako tvorijo super vijačnico. Ti motivi vsebujejo heptadne ponovitve označene (a-b-c-d-e-f-g)n , vsaka dva zavoja vijačnice, "a" in "d" običajno predstavljata nepolame, hidrofobne aminokislinske ostanke, ki se nahajajo na vmesni površini med vijačnicama, "e" in "g" so polarni aminokislinski ostanki, ki so izpostavljeni topilu in interagirajo elektrostatsko, "b", "c" in "f" pa so hidrofilni in izpostavljeni topilu. Različni aminokislinski ostanki na mestih "a-g" določajo oligomerizacijsko stanje, specifičnost, orientacijo vijačnice in stabilnost. Bolj specifično, se izraz "segment za tvorbo ovitih vijačnic" v opisu nanaša na naravne ali načrtovane proteinske strukturne motive, ki tvorijo ovite vijačnice in vsebujejo vsaj dve heptadi in so lahko paraleleni ali antiparalelni in lahko tvorijo homo- ali heterooligomere.The term " wrapped helix segment " has a general meaning and refers to structural protein motifs composed of 2 or more α-helices that wrap around each other and thus form a super helix. These motifs contain heptadic repetitions marked (a-b-c-d-e-f-g) n, every two turns of the helix, " a " in " d " they usually represent non-polar, hydrophobic amino acid residues located on the intermediate surface between the helices, " e " in " g " are polar amino acid residues that are exposed to the solvent and interact electrostatically, " b ", " c " in " f " however, they are hydrophilic and solvent exposed. Various amino acid residues at " a-g " determine the oligomerization state, specificity, helix orientation, and stability. More specifically, the term " wrapped helix segment " in the description, it refers to natural or engineered protein structural motifs that form enveloped helices and contain at least two heptads and may be parallel or antiparallel and may form homo- or heterooligomers.
Segmente za tvorbo ovitih vijačnic lahko izberemo iz naravnih ali načrtovanih proteinskih strukturnih motivov za tvorbo ovitih vijačnic prednostno iz naravnih proteinov z levcinsko zadrgo, kot npr. BZIP ali načrtovanih zaporedij za tvorbo ovitih vijačnic, prednostno izmed: SEQ ID: 14 ali SEQ ID: 26 ali parov SEQ ID: 10 in SEQ ID: 12, SEQ ID: 28 in SEQ ID: 30; SEQ ID:32 in SEQ ID: 34, SEQ ID: 36 in SEQ ID: 38.The segments for the formation of wrapped helices can be selected from natural or planned protein structural motifs for the formation of wrapped helices, preferably from natural proteins with a leucine zipper, such as e.g. BZIP or planned sequences for forming wrapped helices, preferably between: 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.
Funkcionalna polipeptidna domenaFunctional polypeptide domain
Izum se nanaša na odkritje, da ima polipeptidni material lahko poleg zaradi strukturne vloge pomembnih domen za tvorbo ovitih vijačnic in elastinu podobnih domen dodatno vključeno vsaj eno funkcionalno polipeptidno domeno, kar daje polipeptidnemu materialu dodatne -10- funkcionalne lastnosti, kot je na primer spodbujanje rasti celic in njihove diferenciacije, zaviranje rasti patogenov ali vezava kovinskih ionov.The invention relates to the discovery that a polypeptide material may additionally include at least one functional polypeptide domain in addition to the structural role of important domains for the formation of coiled helix and elastin-like domains, which gives the polypeptide material additional -10- functional properties, such as growth promotion cells and their differentiation, inhibition of pathogen growth or binding of metal ions.
Funkcionalna polipeptidna domena je lahko kovalentno vezana na vsaj en elastinu podoben segment z vsaj dvema segmentoma za tvorbo ovitih vijačnic, ki sestavljajo polipeptidni material.The functional polypeptide domain may be covalently linked to at least one elastin-like segment with at least two segments to form the coiled helices that make up the polypeptide material.
Poleg kovalentne vezave funkcionalne domene na polipeptidni material kot je opisano zgoraj, se izum nanaša tudi na drugo možnost vključitve funkcionalne polipeptidne domene v polipeptidni material. Izumitelji so odkrili, da je koristno, če lahko uvedemo funkcionalne lastnosti polipeptidnemu materialu, ki ne vsebuje nobene funkcionalne domene. Izumitelji so odkrili, da polipeptidnemu materialu lahko dodamo funkcionalno polipeptidno domeno, ki je združena s segmentom za tvorbo ovitih vijačnic in tako se funkcionalna polipeptidna domena, združena s segmentom za tvorbo ovitih vijačnic vključi v polipeptidni material preko oligomerizacije komplementarnih segmentov za tvorbo ovitih vijačnic v polipeptidnem materialu.In addition to the covalent binding of a functional domain to a polypeptide material as described above, the invention also relates to another possibility of incorporating a functional polypeptide domain into a polypeptide material. The inventors have found it useful to be able to introduce functional properties to a polypeptide material that does not contain any functional domain. The inventors have discovered that a functional polypeptide domain can be added to a polypeptide material associated with a wrapped helix segment, and thus the functional polypeptide domain joined to the wrapped helix segment is incorporated into the polypeptide material through oligomerization of complementary wrapped segments. material.
Na ta način lahko dodamo polipeptidnemu materialu, ki že vsebuje neko funkcionalno polipeptidno domeno, dodatne zaželene lastnosti z dodajanjem neke druge funkcionalne polipeptidne domene, združene s segmentom za tvorbo ovitih vijačnic, ki je komplementaren segmentu za tvorbo ovitih vijačnic v polipeptidnem materialu. Funkcionalne polipeptidne domene je mogoče dodati polipeptidnemu materialu ob začetnem sestavljanju ali naknadno in poleg tega jih lahko vnesemo v organizem na različnih mestih, vendar z njimi še vedno ciljamo na mesto vsajenega polipeptidnega materiala s komplementarnimi segmenti za tvorbo ovitih vijačnic.In this way, additional desirable properties can be added to a polypeptide material already containing a functional polypeptide domain by adding another functional polypeptide domain combined with a helix-forming helix segment that is complementary to the helix-forming helix segment in the polypeptide material. Functional polypeptide domains can be added to the polypeptide material at initial or subsequent assembly and can be introduced into the body at various sites, but they are still targeted to the site of the implanted polypeptide material with complementary segments to form coiled helices.
Kadar je polipeptidna funkcionalna domena kovalentno vezana na polipeptidni material, je dostopna celicam, tkivu oz. organu. Lokalna koncentracija funkcionalne polipeptidne domene je višja kot v primeru topne funkcionalne polipeptidne domene zaradi odsotnosti difuzije. Poleg tega je potrebno manj funkcionalne polipeptidne domene za dosego željenega lokalnega učinka kot v primeru, da je domena v topni obliki. Poleg tega so na ta način možna toksičnost in škodljivi učinki omejeni na področje vsajenega polipeptidnega materiala. Takšen tip materiala tudi omogoča časovno spreminjanje terapije z zaporednim vnašanjem različnih funkcionalnih proteinov. -11- • ····When a polypeptide functional domain is covalently linked to a polypeptide material, it is accessible to cells, tissue, or organ. The local concentration of the functional polypeptide domain is higher than in the case of the soluble functional polypeptide domain due to the absence of diffusion. In addition, fewer functional polypeptide domains are required to achieve the desired local effect than if the domain is in soluble form. In addition, in this way, potential toxicity and adverse effects are limited to the area of the implanted polypeptide material. This type of material also allows for time-varying therapy with sequential administration of different functional proteins. -11- • ····
Izraz "združen (fuzijski) ali fuzija" ima splošen pomen, opis pa se nanaša na proces priprave hibridnega proteina iz dveh predhodno ločenih proteinov/domen/segmentov, izbirno povezanih s povezovalnim delom, ki vsebuje od ene do 20 aminokislin.The term " merged or fusion " has a general meaning, and the description refers to the process of preparing a hybrid protein from two previously separated proteins / domains / segments optionally linked to a linker containing from one to 20 amino acids.
Izraz "kovalentno vezan" ima splošni pomen in se nanaša na funkcionalne polipeptidne domene, ki so na polipeptidni material vezane s peptidno vezjo.The term " covalently bound " has a general meaning and refers to functional polypeptide domains that are linked to the polypeptide material by a peptide bond.
Izraz "komplementaren" se nanaša na sposobnost enega segmenta za tvorbo ovitih vijačnic, da homo- oziroma heterooligomerizira z drugim segmentom za tvorbo ovitih vijačnicThe term " complementary " refers to the ability of one segment to form wrapped helices to homo- or heterooligomerize with another segment to form wrapped helices
Izraz "funkcionalna polipeptidna domena" se nanaša na molekule, ki v polipeptidni material vnašajo neke funkcionalne lastnosti, kot je na primer spodbujanje rasti celic, inhibicija namnoževanja bakterij, diferenciacija celic ali vezava kovinskih ionov. Funkcionalna polipeptidna domena lahko spada med, vendar ni omejena na: rastne faktorje, protimikrobne peptide in domene za vezavo kovinskih ionov.The term " functional polypeptide domain " refers to molecules that impart certain functional properties to a polypeptide material, such as promoting cell growth, inhibiting bacterial proliferation, cell differentiation, or binding metal ions. The functional polypeptide domain may include, but is not limited to: growth factors, antimicrobial peptides, and metal ion binding domains.
Izraz "rastni faktor" ima splošni pomen in se nanaša na molekulo, ki se veže na celične receptorje in uravnava rast, razmnoževanje ali diferenciacijo tarčnih celic ali tkiv. Primeri rastnih faktorjev vključujejo, vendar niso omejeni na: epidermalni rastni faktor (EGF), fibroblastni rastni faktor, živčni rastni faktor (NGF), eritropoetin in druge; prednostno NGF, prednostno SEQ ID NO:. 24.The term " growth factor " has a general meaning and refers to a molecule that binds to cellular receptors and regulates the growth, proliferation or differentiation of target cells or tissues. Examples of growth factors include, but are not limited to: epidermal growth factor (EGF), fibroblast growth factor, nerve growth factor (NGF), erythropoietin, and others; preferably NGF, preferably SEQ ID NO :. 24.
Izraz "protimikrobni peptid" se nanaša na širok spekter antibiotikov, ki delujejo baktericidno, lahko pa delujejo tudi na glive ali viruse s povzročanjem poškodb na membrani, vpletanjem v metabolizem ali ciljanjem citoplazemskih komponent. Primeri protimikrobnih peptidov vključujejo, vendar niso omejeni na: katelicidine in defenzine; prednostno katelicidin LL-37, prednostno SEQ ID NO: 16.The term " antimicrobial peptide " refers to a wide range of antibiotics that act bactericidal, but can also act on fungi or viruses by causing membrane damage, interfering with metabolism, or targeting cytoplasmic components. Examples of antimicrobial peptides include, but are not limited to: cathelicidins and defensins; preferably cathelicidin LL-37, preferably SEQ ID NO: 16.
Izraz "domene za vezavo kovin" ima splošni pomen in se nanaša na peptide, ki lahko vežejo kovine vključno z, vendar ne omejeno na, Ag, Au, Pd, Pt, kar ima protimikrobni učinek. Domena za vezavo kovin je prednostno SEQ ID NO: 26, ki je sestavljena iz 3 odsekov zmožnih trimerizacije in tvori srebrne nanodelce.The term " metal binding domain " has a general meaning and refers to peptides that can bind metals including, but not limited to, Ag, Au, Pd, Pt, which has an antimicrobial effect. The metal binding domain is preferably SEQ ID NO: 26, which consists of 3 sections capable of trimerization and forms silver nanoparticles.
Izum vključuje tudi spoznanje, da je polipeptidni material, ki vsebuje tudi funkcionalno polipeptidno domeno, kovalentno vezano na polipeptidni material, lahko tudi mešanica (i) polipeptidnega materiala, ki vsebuje vsaj en elastinu podoben segment in vsaj dva segmenta za tvorbo ovitih vijačnic, in (ii) polipeptidnega materiala, ki poleg vsaj enega elastinu podobnega segmenta in vsaj dveh segmentov za tvorbo ovitih vijačnic vsebuje tudi funkcionalno polipeptidno domeno.The invention also includes the recognition that a polypeptide material that also contains a functional polypeptide domain covalently linked to the polypeptide material may also be a mixture of (i) a polypeptide material comprising at least one elastin-like segment and at least two segments for forming wrapped helices, and ( ii) a polypeptide material comprising a functional polypeptide domain in addition to at least one elastin-like segment and at least two segments for forming wrapped helices.
Glede na zaželene lastnosti polipeptidnega materiala, je lahko mešanica sestavljena iz različnih razmerij, pri čemer polipeptidni material, ki poleg vsaj enega elastinu podobnega segmenta in vsaj dveh segmentov za tvorbo ovitih vijačnic vsebuje tudi funkcionalno polipeptidno domeno, predstavlja manj kot 50 % mešanice, bolj značilno 20 % in najpogosteje 1-5 % mešanice. Polipeptidni materiali so lahko zmešani na osnovi molamosti, mase, volumna, ipd.Depending on the desired properties of the polypeptide material, the mixture may consist of different proportions, wherein the polypeptide material, which in addition to at least one elastin-like segment and at least two segments for forming wrapped helices also contains a functional polypeptide domain, represents less than 50% of the mixture. 20% and most often 1-5% of the mixture. Polypeptide materials may be mixed on the basis of molarity, mass, volume, and the like.
Takšne heterogene mešanice imajo določene prednosti pred homogenimi. Homogene mešanice namreč vsebujejo le polipeptidni material, ki vsebuje vsaj en elastinu podoben segment in vsaj dva segmenta za tvorbo ovitih vijačnic, kar ne zagotavlja željenih lastnosti, kot so pospeševanja rasti celic, celične adhezije, zaviranje rasti bakterij, diferenciacije celic, vezave kovinskih ionov, ipd. Po drugi strani pa homogene mešanice, ki vsebujejo samo polipeptidni material, ki poleg vsaj enega elastinu podobnega segmenta in vsaj dveh segmentov za tvorbo ovitih vijačnic vsebuje tudi funkcionalno polipeptidno domeno, tvorijo material, kije šibkejši v primerjavi s polipeptidnim materialom, ki je sestavljen iz vsaj enega elastinu podobnega segmenta in vsaj dveh segmentov za tvorbo ovitih vijačnic.Such heterogeneous mixtures have certain advantages over homogeneous ones. Homogeneous mixtures contain only a polypeptide material containing at least one elastin-like segment and at least two segments for the formation of wrapped helices, which does not provide the desired properties such as cell growth promotion, cell adhesion, bacterial growth inhibition, cell differentiation, metal ion binding, etc. On the other hand, homogeneous mixtures containing only a polypeptide material which, in addition to at least one elastin-like segment and at least two segments for forming wrapped helices, also contain a functional polypeptide domain, form a material that is weaker than a polypeptide material consisting of at least one elastin-like segment and at least two segments to form wrapped helices.
Da bi združili željene lastnosti polipeptidnih materialov, ki so zgrajeni iz različnih pripadajočih funkcionalnih polipeptidnih domen, izum vključuje tudi mešanje večih polipeptidnih materialov z različnimi pripadajočimi funkcionalnimi domenami s polipeptidnimi materiali, ki ne vsebujejo funkcionalnih domen.In order to combine the desired properties of polypeptide materials constructed from different associated functional polypeptide domains, the invention also includes mixing multiple polypeptide materials with different associated functional domains with polypeptide materials that do not contain functional domains.
Polipeptidni material, ki ga je mogoče razstaviti z uporabo peptida, ki vsebuje segment za tvorbo ovitih vijačnicA polypeptide material that can be disassembled using a peptide containing a segment to form coiled helices
Osnova izuma je prav tako odkritje, da je polipeptidni material možno razstaviti z dodatkom peptida, ki vključuje segment za tvorbo voitih vijačnic in je prav tako prisoten v polipeptidnem materialu.The basis of the invention is also the discovery that the polypeptide material can be disassembled by the addition of a peptide that includes a segment for forming helices and is also present in the polypeptide material.
Izumitelji so odkrili, da dodatek peptida, ki vključuje segment za tvorbo ovitih vijačnic in je podoben segmentu za tvorbo ovitih vijačnic v polipeptidnem materialu predstavlja pristop, s katerim dosežemo, da se polipeptidni material razgradi. Dodan segment za tvorbo ovitih vijačnic se vključi v strukturo polipeptidnega materiala zaradi sposobnosti tvorbe oligomerov z -13- enim od segmentov za tvorbo ovitih vijačnic v polipeptidnem materialu, zaradi česar se polipeptidni material zrahlja, kar vodi v njegovo razgradnjo.The inventors have discovered that the addition of a peptide that includes a segment for the formation of enveloped helices and is similar to the segment for the formation of enveloped helices in the polypeptide material represents an approach by which the polypeptide material is degraded. An added helix formation segment is incorporated into the structure of the polypeptide material due to its ability to form oligomers with -13- one of the helix formation segments in the polypeptide material, causing the polypeptide material to loosen, leading to its degradation.
Na ta način lahko ločimo celice, ki rastejo na polipeptidnem materialu od omenjenega materiala, brez da bi posegali po kemijskih spremembah (npr. pH, ionska jakost), fizikalnih spremembah (npr. dolge inkubacije pri visokih temperaturah, osmotski šok) ali encimatski razgradnji polipeptidnega materiala, ki lahko vplivajo na ali poškodujejo površinske receptorje in adhezijske molekule ali povzročijo stres celicam. Tako pridobljene celice se lahko prešteje, presadi naprej ali uporabi za nadaljnjo biokemično analizo ali tehnološke aplikacije.In this way, cells growing on the polypeptide material can be separated from said material without interfering with chemical changes (eg pH, ionic strength), physical changes (eg long incubations at high temperatures, osmotic shock) or enzymatic degradation of the polypeptide. material that can affect or damage surface receptors and adhesion molecules or cause stress to cells. The cells thus obtained can be counted, transplanted or used for further biochemical analysis or technological applications.
Izraz " peptid, ki vključuje segment, za tvorbo ovitih vijačnic" v opisu se nanaša na segment, za vtorbo ovitih vijačnic, pri čemer je segment zmožen oligomerizacije z enim od segmentov za tvorbo ovitih vijačnic v polipeptidnem materialu. Izraz " segment za tvorbo ovitih vijačnic" se nanaša na segmente za tvorbo ovitih vijačnic in so opisani zgoraj.The expression " a peptide comprising a segment to form coiled helices " in the description, it refers to a segment for inserting wrapped helices, wherein the segment is capable of oligomerization with one of the segments to form wrapped helices in the polypeptide material. The expression " wrapped helix segment " refers to the segments for the formation of wrapped helices and are described above.
Polipeptidni material sestavljen iz vsaj dveh različnih polipeptidnih materialovA polypeptide material composed of at least two different polypeptide materials
Osnova izuma je tudi odkritje polipeptidnega materiala, ki je kombinacija vsaj dveh različnih polipeptidnih materialov. Sestavljen polipeptidni material je pripravljen s kombiniranjem dveh polipeptidnih materialov, pri čemer segmenti za tvorbo ovitih vijačnic iz enega od materialov lahko tvorijo heterooligomere s segmenti za tvorbo ovitih vijačnic v drugem materialu. Izumitelji so odkrili, da heterooligomerizacija segmentov za tvorbo ovitih vijačnic iz enega materiala s segmenti za tvorbo ovitih vijačnic iz drugega materiala, predstavlja način uravnavanja začetka sestavljanja polipeptidnega materiala. Topni polipeptidni material, ki vsebuje segment za tvorbo ovitih vijačnic in je sposoben heterooligomerizacije, se namreč ne sestavi, če predstavlja edini polipeptidni material v raztopini. Tvorbo polipeptidnega materiala je mogoče sprožiti šele z dodatkom drugega materiala, ki vsebuje segment za tvorbo ovitih vijačnic in heterooligomerizira s segmentom za tvorbo ovitih vijačnic iz prvega materiala , kar predstavlja mehanizem za nadzorovano sestavljanje materiala.The invention is also based on the discovery of a polypeptide material which is a combination of at least two different polypeptide materials. The composite polypeptide material is prepared by combining two polypeptide materials, wherein the helix-forming helix segments of one of the materials can form heterooligomers with the helix-forming helix segments in the other material. The inventors have discovered that heterooligomerization of segments for forming wrapped helices from one material with segments for forming wrapped helices from another material represents a way of regulating the onset of assembly of the polypeptide material. Namely, a soluble polypeptide material that contains a segment for the formation of coiled helices and is capable of heterooligomerization is not assembled if it is the only polypeptide material in solution. The formation of the polypeptide material can only be initiated by the addition of a second material containing a wrapped helix forming segment and heterooligomerized with the wrapped helix forming segment from the first material, which is a mechanism for controlled material assembly.
Sestavljeni material vsebuje vsaj dva polipeptidna materiala, lahko pa je tudi sestavljen iz večih, tudi do deset, bolj pogosto iz dveh do osmih različnih polipeptidnih materialov, ki se nanašajo na izum. -14-The composite material contains at least two polypeptide materials, but may also consist of several, even up to ten, more often of two to eight different polypeptide materials related to the invention. -14-
Protein, ki sestavlja polipeptidni materialA protein that makes up a polypeptide material
Opis se nanaša na protein, ki sestavlja polipeptidni material opisan zgoraj. Protein vsebuje vsaj en elastinu podoben segment in vsaj dva segmenta za tvorbo ovitih vijačnic, pri čemer se vsaj eden elastinu podoben segment nahaja med dvema ali več segmenti za tvorbo ovitih vijačnic in pri čemer so posamezne polipeptidne molekule ned sabo povezane preko interakcij med segmenti za tvorbo ovitih vijačnic na teh molekulah. Poleg tega lahko protein, ki sestavlja polipeptidni material, vsebuje tudi funkcionalno polipeptidno domeno, ki je izbirno lahko del proteina. Kadar je funkcionalna polipeptidna domena del proteina, ki sestavlja polipeptidni material, je ta kovalentno vezana in ne more difundirati stran, kar predstavlja pomembno prednost.The description refers to the protein that makes up the polypeptide material described above. The protein comprises at least one elastin-like segment and at least two segments for the formation of enveloped helices, wherein the at least one elastin-like segment is located between two or more segments for the formation of enveloped helices, and the individual polypeptide molecules are inextricably linked through interactions wrapped helices on these molecules. In addition, the protein that makes up the polypeptide material may also contain a functional polypeptide domain that may optionally be part of the protein. When the functional polypeptide domain is part of the protein that makes up the polypeptide material, it is covalently bound and cannot diffuse away, which is an important advantage.
Izum se nanaša na protein, ki vsebuje združene segmente za tvorbo ovitih vijačnic in funkcionalno polipeptidno domeno. Izumitelji so prišli do odkritja, da je funkcionalno polipeptidno domeno mogoče vključiti v polipeptidni material preko interakcij segmentov za tvorbo ovitih vijačnic s komplementarnimi segmenti za tvorbo ovitih vijačnic, ki se nahajajo v materialu. Na ta način materialu lahko dodamo funkcionalne lastnosti.The invention relates to a protein comprising fused segments for the formation of enveloped helices and a functional polypeptide domain. The inventors have discovered that a functional polypeptide domain can be incorporated into a polypeptide material through the interactions of wrapped helix segments with complementary helix forming segments present in the material. In this way, functional properties can be added to the material.
Segmenti in domene proteina opisanega zgoraj so lahko izbirno ločene ena od druge s povezovalnim delom, ki vsebuje eno do 20 aminokislin, prednostno eno do 6 aminokislin. Proteini lahko izbirno vsebujejo signalno sekvenco, ki usmerja izločanje proteina in aminokislinske označevalce za čiščenje in detekcijo proteina.The segments and domains of the protein described above may be optionally separated from each other by a binding portion containing one to 20 amino acids, preferably one to 6 amino acids. Proteins may optionally contain a signal sequence that directs protein secretion and amino acid markers for protein purification and detection.
Izraz "povezovalni del" v opisu se nanaša na krajša aminokislinska zaporedja, katerih naloga je le ločevanje posameznih domen oz. segmentov proteina. Vloga povezovalnega dela, ki se ga lahko izbirno vključi v protein, , je lahko tudi vnos cepitvenih mest za posttranslacijske modifikacije, vključno z vnosom mest za boljše procesiranje. Dolžina povezovalnega dela ni omejena, čeprav je ponavadi dolg do 30 aminokislin, prednostno eno do 20 aminokislin, najpogosteje eno do 6 aminokislin. V povezovalni del je lahko vključena katerakoli aminokislina, prednostno pa so aminokisline izbrane izmed, vendar ne omejene na: serinske, glicinske, treoninske, prolinske, valinske in alaninske ostanke, ki omogočajo fleksibilnost in specifično intermolekulamo asociacijo le preko segmentov za tvorbo ovitih vijačnic ali preko elastinu podobnih domen.The term " connecting part " in the description it refers to shorter amino acid sequences, whose task is only to separate individual domains or. protein segments. The role of the binding portion, which may optionally be incorporated into the protein, may also be the insertion of cleavage sites for posttranslational modifications, including the insertion of sites for better processing. The length of the linker is not limited, although it is usually up to 30 amino acids long, preferably one to 20 amino acids, most often one to 6 amino acids. Any amino acid may be included in the linking moiety, preferably amino acids selected from, but not limited to: serine, glycine, threonine, proline, valine and alanine residues that allow flexibility and specific intermolecular association only through segments for the formation of enveloped helices or via elastin-like domains.
Izraz "signalno zaporedje" v opisu se nanaša na aminokislinsko zaporedje, ki je pomembno za usmerjanje proteina na določeno mesto v celici. Signalno zaporedje je odvisno od -15- gostiteljskega organizma v katerem se fuzijski protein izraža. Aminokislinsko zaporedje signalnih zaporedij so dobro poznani strokovnjakom, prav tako tudi katero signalno zaporedje je funkcionalno v določenem organizmu.The term " signal sequence " in the description, it refers to an amino acid sequence that is important for directing a protein to a specific site in a cell. The signal sequence depends on the -15- host organism in which the fusion protein is expressed. The amino acid sequence of signal sequences is well known to those skilled in the art, as is which signal sequence is functional in a particular organism.
Izraz "aminokislinski označevalec/ci" se nanaša na zaporedja aminokislin, ki so dodane proteinu z namenom olajšanja čiščenja, izolacije ali detekcije proteina.The term " amino acid marker (s) " refers to the amino acid sequences added to a protein for the purpose of facilitating the purification, isolation, or detection of the protein.
Mesto signalnega zaporedja, povezovalnih delov in aminokislinskih označevalcev je izbirno, vendar mora omogočiti funkcionalno izražanje proteina in ohraniti funkcijo, zaradi katere so bila ta aminokislinska zaporedja izbrana. DNK, ki zapisuje protein polipeptidnega materialaThe location of the signal sequence, linkers, and amino acid markers is optional, but it must allow for the functional expression of the protein and maintain the function for which those amino acid sequences were selected. DNA that records the protein of a polypeptide material
Izum se nanaša tudi na DNK, ki zapisuje protein polipeptidnega materiala, ki vsebuje vsaj en elastinu podoben segment in vsaj dva segmenta za tvorbo ovitih vijačnic, pri čemer se vsaj en elastinu podoben segment nahaja med dvema ali več segmenti za tvorbo ovitih vijačnic in pri čemer se posamezne polipeptidne molekule povezujejo med sabo preko interakcij med segmenti za tvorbo ovitih vijačnic na teh molekulah, in so izbirno povezani s funkcionalno proteinsko domeno, segmenti oz. domene polipeptida pa so druga z drugo izbirno povezani/e s povezovalnim delom. Protein izbirno vsebuje signalno zaporedje in aminokislinske označevalce. Opis se nanaša tudi na DNK, ki zapisuje protein polipeptidnega materiala, ki vsebuje združene segmente za tvorbo ovitih vijačnic in funkcionalne polipeptidne domene, pri čemer so segmenti in domene izbirno povezani drug z drugim s povezovalnim delom, protein pa izbirno vsebuje tudi signalna zaporedja in aminokislinske označevalce.The invention also relates to DNA that records a protein of a polypeptide material comprising at least one elastin-like segment and at least two segments for forming wrapped helices, wherein the at least one elastin-like segment is located between two or more segments for forming wrapped helices, and wherein individual polypeptide molecules bind to each other through interactions between segments to form coiled helices on these molecules, and are optionally linked to a functional protein domain, segments or the polypeptide domains are optionally linked to each other by a binding member. The protein optionally contains a signal sequence and amino acid markers. The description also relates to DNA that records a protein of a polypeptide material comprising fused helix-forming segments and a functional polypeptide domain, wherein the segments and domains are optionally linked to each other by a binding moiety, and the protein optionally contains signal sequences and amino acid sequences. markers.
Izraz "DNK" v opisu se nanaša na zaporedje nukleotidov v odprtem bralnem okvirju, ki zapisuje protein, ki je predmet izuma, in je operativno povezano z regulatomimi elementi, promotorjem in terminatorjem, kar omogoča izražanje proteina v gostiteljskih celicah. Dolžina DNK zaporedja se lahko precej razlikuje, odvisno od posameznega proteina.The term " DNA " in the description refers to a nucleotide sequence in an open reading frame that records the protein of the invention and is operably linked to regulatory elements, a promoter and a terminator, allowing protein to be expressed in host cells. The length of the DNA sequence can vary considerably depending on the individual protein.
Izraz "regulatomi elementi" v opisu ima splošen pomen in se nanaša na regijo DNK v ekspresijskem vektorju, kamor se vežejo regulatomi proteini, kot so npr. transkripcijski faktorji, in uravnavajo izražanje gena.The term " regulatory elements " in the description it has a general meaning and refers to the region of DNA in the expression vector to which regulatory proteins such as e.g. transcription factors, and regulate gene expression.
Izraz "promotor" v opisu ima splošen pomen in se nanaša na regijo DNK v ekspresijskem vektorju, ki omogoča prepis tarčnega gena. Tip promotorja je odvisen od gostiteljskega -16- • · organizma, v katerem bo gen izražen. V primeru izražanja v prokariontih, npr. E.coli, je možna uporaba večih promotorjev, npr. P]ac, Pts , prednostno se uporabi PT7. V primeru izražanja v evkariontih, npr. v sesalskih celicah, so promotorji najpogosteje virusnega izvora, kot npr. Pcmv ali Psv40·The term " promoter " in the description, it has a general meaning and refers to the region of DNA in the expression vector that allows the transcription of the target gene. The type of promoter depends on the host -16- • · organism in which the gene will be expressed. In the case of expression in prokaryotes, e.g. E.coli, it is possible to use several promoters, e.g. P] ac, Pts, preferably PT7 is used. In the case of expression in eukaryotes, e.g. in mammalian cells, promoters are most commonly of viral origin, such as e.g. Pcmv or Psv40 ·
Izraz "terminator" v opisu ima splošni pomen in se nanaša na na zaporedje, ki označuje konec gena za prepis. Tip terminatorja je odvisen od gostiteljskega organizma v katerem bo gen izražen. V primeru izražanja v prokariontih, npr. E.coli, je možna uporaba terminatorja iz bakteriofaga T7.The term " terminator " in the description it has a general meaning and refers to the sequence that marks the end of the transcription gene. The type of terminator depends on the host organism in which the gene will be expressed. In the case of expression in prokaryotes, e.g. E.coli, it is possible to use a terminator from bacteriophage T7.
Rekombinantna nukleinska kislina V izumu so bile uporabljene standardne metode molekularne biologije, ki so splošno znane strokovnjakom na tem področju (glej Sambrook et al. 1989. Molecular Cloning: A laboratory manual, 2nd ed., Cold Spring Harbor, NY, Ausubel et al. Current Protocols in Molecular Biology, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., NY).Recombinant Nucleic Acid The invention used standard methods of molecular biology 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. Current Protocols in Molecular Biology, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., NY).
Opisane proteine za polipeptidni material je mogoče sintetizirati z izražanjem DNK, ki zapisuje te proteine, v primernem gostiteljskem organizmu. DNK, ki zapisuje proteine, se vstavi v ustrezen ekspresijski vektor. Ustrezni vektorji vključujejo, vendar niso omejeni na: plazmide, virusne vektorje, ipd. Ekspresijski vektorji, ki so kompatibilni s celicami gostitelj skega organizma, so dobro poznani strokovnjakom na področju in vsebujejo ustrezne kontrolne elemente za prepis in prevod nukleotidnega zaporedja. Po navadi ekspresijski vektor vključuje ekspresijsko kaseto, ki je v 5' proti 3' smeri sestavljena iz promotorja, kodirajočega zaporedja za fuzijski protein operativno povezanega z regulatomimi elementi, promoterjem in terminatoijem, vključno s stop kodonom za RNA polimerazo in poliadenilacijskim signalom za poliadenilazo.The described proteins for the polypeptide material can be synthesized by expressing the DNA that records these proteins in a suitable host organism. The DNA that writes the proteins is inserted into the appropriate expression vector. Relevant vectors include, but are not limited to: plasmids, viral vectors, and the like. Expression vectors that are compatible with the cells of the host organism are well known to those skilled in the art and contain appropriate control elements for transcription and translation of the nucleotide sequence. Typically, the expression vector includes an expression cassette consisting in a 5 'to 3' direction of a promoter encoding a sequence for a fusion protein operably linked to regulatory elements, a promoter and terminatology, including a stop codon for RNA polymerase and a polyadenylation signal for polyadenylase.
Ekspresijski vektorje lahko pripravljen za izražanje v prokariontskih ali evkariontskih celicah. Primer prokariontskih celic so bakterije, npr. Escherichia coli. V skladu z izumom, se prokarionske celice uporabljajo za pridobivanje zadostne količine nukleinske kisline. Ekspresijski vektor ponavadi vsebuje kontrolne elemente, ki so operativno povezani z DNK izuma, ki nosi zapis za proteine.. Kontrolni elementi so izbrani tako, da sprožijo učinkovito in tkivno specifično izražanje. Promotor je lahko konstitutiven ali inducibilen, odvisno od željenega vzorca izražanja. Promotor lahko izvira iz gostiteljskega organizma ali pa je tujega -17- • · izvora (se ne nahaja v celicah, kjer ga uporabljamo), lahko je naraven ali sintetičen. Promotor mora biti izbran tako, da deluje v tarčnih celicah gostiteljskega organizma. Poleg tega so vključeni tudi signali za začetek in učinkovito traslacijo fuzijskega proteina - ATG in pripadajoča zaporedja. Kadar vektor, uporabljen v tem izumu, vključuje dva ali več bralnih okvirjev, morajo biti ti neodvisno povezani s kontrolnimi elementi, kontrolni elementi pa so lahko enaki ali pa različni, odvisno od željene produkcije proteinov.Expression vectors may be ready 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 typically contains control elements that are operably linked to the DNA of the invention that carries the protein transcript. The control elements are selected to trigger efficient and tissue-specific expression. The promoter may be constitutive or inducible, depending on the desired expression pattern. The promoter may originate from the host organism or be of foreign origin (not found in the cells where it is used), it may be natural or synthetic. The promoter must be selected to act in the target cells of the host organism. In addition, signals for the initiation and efficient translation of the fusion protein - ATG and associated sequences are also included. When the vector used in the present invention includes two or more reading frames, these must be independently linked to control elements, and the control elements may be the same or different, depending on the desired protein production.
Primeri bakterijskih ekspresijskih vektorjev vključujejo, vendar niso omejeni na: pET vektorje, pRSET vektorje, in druge. Kadar so vektorji uporabljeni v bakterijskih celicah, so kontrolni elementi bakterijskega izvora.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.
Primeri sesalskih ekspresijskih vektorjev za sesalske celice vključujejo, vendar niso omejeni na: pcDNA (Invitrogen), pFLAG (Sigma), in druge. Kadar so vektorji uporabljeni v sesalskih celicah, so kontrolni elementi v večini primerov virusnega izvora, iz npr. adenovirusa 2, citomegalovirusa, virusa Simian virus 40.Examples of mammalian expression vectors for mammalian cells include, but are not limited to: pcDNA (Invitrogen), pFLAG (Sigma), and others. When vectors are used in mammalian cells, the control elements are in most cases of viral origin, from e.g. adenovirus 2, cytomegalovirus, Simian virus 40.
Gostiteljski organizemHost organism
Izum se nanaša tudi na gostiteljski organizem, ki izraža zgoraj opisane proteine.The invention also relates to a host organism expressing the proteins described above.
Izraz "gostiteljski organizem" se nanaša na organizem, v katerega je bila vnešena DNK, ki zapisuje protein, z namenom, da se tam izrazi. Prenos vektorjev v gostiteljske organizme poteka po konvencionalnih metodah, ki so znane strokovnjakom na področju; to so: transformacija, transfekcija, vključno z: kemično transformacijo, elektroporacijo, mikroinjekcijo, DNK lipofekcijo, soniciranjem celic, mikrobombandiranjem, virusnim vnosom DNK in drugimi. V skladu z izumom je vnos DNK s transformacijo v bakterijske celice.The term " host organism " refers to the organism into which the DNA that records the protein has been introduced in order to express it there. The transfer of vectors to host organisms is carried out by conventional methods known to those skilled in the art; these are: transformation, transfection, including: chemical transformation, electroporation, microinjection, DNA lipofection, cell sonication, microbombing, viral DNA uptake and others. According to the invention, the uptake of DNA is by transformation into bacterial cells.
Gostiteljski organizrm je lahko pro- ali evkariontski. Evkariontske celice, primerne za izražanje fuzijskega proteina, , niso omejene za uporabo, dokler so celične linije kompatibilne z metodami propagacije ekspresij skega vektorja in z izražanjem fuzijskega proteina. Prednostne evkariontske celice vključujejo, vendar niso omejene na: kvasovke, glive, rastlinske celice in sesalske celice, kot so: mišji, podganji, opičji ali človeški fibroblasti.The host organism may be pro- or eukaryotic. Eukaryotic cells suitable for fusion protein expression are not restricted for use as long as the cell lines are compatible with expression vector propagation methods and fusion protein expression. Preferred eukaryotic cells include, but are not limited to: yeast, fungi, plant cells, and mammalian cells, such as: mouse, rat, monkey, or human fibroblasts.
Za izražanje DNK se lahko v skladu z izumom uporabi katerikoli bakterijski gostitelj. Za izražanje proteinov tega izuma, je najbolj priporočljiva uporaba bakterij ali kvasovk. Protein se lahko izraža v bakterijah E. coli ali B. subtilis ali v kvasovkah S. cerevisiae ali P. pastoris.Any bacterial host can be used for DNA expression according to the invention. To express the proteins of the present invention, the use of bacteria or yeasts is most preferred. The protein can be expressed in E. coli or B. subtilis bacteria or in S. cerevisiae or P. pastoris yeasts.
Preferenčna bakterija je E. coli. Izum se nanaša na bakterije ali kvasovke, ki izražajo protein, prednostno na bakterije E. coil ali kvasovke P. pastoris.The preferred bacterium is E. coli. The invention relates to bacteria or yeasts expressing a protein, preferably to E. coil bacteria or P. pastoris yeasts.
Produkcija proteina/proces za pripravo polipeptidnega materialaProtein production / process for preparation of polypeptide material
Izum se nanaša tudi na proces proizvajanja polipeptidnega materiala in vključuje uvajanje DNK, ki zapisuje protein polipeptidnega materiala v gostiteljski organizem, gojenje gostiteljskega organizma pri pogojih primernih za ekspresijo proteina, izolacijo proteina in izpostavljanje proteina specifičnim pogojem z namenom tvorbe polipeptidnega materiala.The invention also relates to a process for producing a polypeptide material and includes introducing DNA that records the protein polypeptide material into a host organism, culturing the host organism under conditions suitable for protein expression, protein isolation and exposing the protein to specific conditions to form the polypeptide material.
Fuzijski protein je mogoče sintetizirati v gostiteljskem organizmu,, ki izraža heterologno nukleinsko kislino, ki zapisuje fuzijski protein. Fuzijski protein tega izuma se uporabi za pripravo polipeptidnega materiala. Fuzijski protein je lahko povezan s signalnim zaporedjem, kije kodiran v nukleinski kislini. V splošnem je heterologna nukleinska kislina vključena v ekspresijski vektor (virusni ali ne-virusni), ki je opisan zgoraj.The fusion protein can be synthesized in a host organism that expresses a heterologous nucleic acid that records the fusion protein. The fusion protein of the present invention is used to prepare a polypeptide material. The fusion protein may be associated with a signal sequence encoded in the nucleic acid. In general, a heterologous nucleic acid is included in the expression vector (viral or non-viral) described above.
Izum zajema gostiteljske celice ali organizme, ki vsebujejo nukleinsko kislino (prehodno ali stabilno), ki nosi zapis za fuzijske proteine opisane zgoraj. Primerni gostiteljski organizmi so znani izvedencem na področju molekularne biologije in vključujejo bakterijske in evkariotske celice.The invention encompasses host cells or organisms containing a nucleic acid (transient or stable) bearing the transcript for the fusion proteins described above. Suitable host organisms are known to those skilled in the art of molecular biology and include bacterial and eukaryotic cells.
Vnos vektorjev v gostitelj ske celice je izpeljan po konvencionalnih, poznanih metodah, kot je opisano zgoraj.The introduction of vectors into the host cell is performed according to conventional, known methods, as described above.
Vnos DNK je lahko prehoden ali stabilen. Prehodno izražanje se nanaša na vnos DNK vektorja, ki se ne vgradi v genom celice. Stabilen vnos se doseže z vnosom DNKv gostiteljev genom. Prenos DNK, še posebno pri pripravi gostiteljskega organizma s stabilno integracijo DNK, je možno nadzororvati s prisotnostjo markerjev. DNK, ki zapisuje markerje, se nanaša na odpornost na snovi, npr. antibiotike, in je lahko vključena v vektor, ki vsebuje gene za fuzijske proteine, ali v ločen vektor.DNA uptake can be transient or stable. Transient expression refers to the entry of a DNA vector that is not incorporated into the genome of a cell. Stable uptake is achieved by uptake of DNA into the host genome. DNA transfer, especially in the preparation of the host organism with stable DNA integration, can be controlled by the presence of markers. DNA that writes markers refers to resistance to substances, e.g. antibiotics, and may be included in a vector containing genes for fusion proteins, or in a separate vector.
Protein je izražen v ustreznem gostiteljskem organizmu. Ekspresijo velikih količin fuzijskega proteina lahko dosežemo pri bakterijah, kot je E. coli, in pri kvasovkah P. pastoris', poleg tega je možna tudi uporaba kultur sesalskih celic za produkcijo manjših količin proteina, kadar so posttranslacijske modifikacije pomembne za pravilno zlaganje in funkcijo. Znano je, da je -19-The protein is expressed in the corresponding host organism. Expression of large amounts of fusion protein can be achieved in bacteria such as E. coli and P. pastoris' yeasts, and mammalian cell cultures can be used to produce smaller amounts of protein when posttranslational modifications are important for proper folding and function. It is known that -19-
mogoče izraziti protein v celicah naslednjih organizmov: človeka, glodalcev, goveda, svinj, perutnine, zajcev ipd. Gostiteljske celice lahko gojimo kot primarne ali kot nesmrtne linije. Izražanje fuzijskih proteinov je lahko konstitutivno ali inducibilno, npr. izražanje proteina lahko sproži dodatek induktorja IPTG, ki zaradi vezave represorja lac sproži ekspresijo želenega proteina. Fuzijski protein je lahko v topni frakciji ali pa v inkluzijskih telescih.protein can be expressed in the cells of the following organisms: humans, rodents, cattle, pigs, poultry, rabbits, etc. Host cells can be grown as primary or as immortal lines. The expression of fusion proteins can be constitutive or inducible, e.g. protein expression can be triggered by the addition of an IPTG inducer, which triggers the expression of the desired protein due to binding of the lac repressor. The fusion protein may be in a soluble fraction or in inclusion bodies.
Tehnike čiščenja proteina vključujejo kromatografijo z ločevanjem po velikosti, ionsko izmenjevalno kromatografijo, kromatografijo z reverzno fazo in afinitetno kromatografijo, kadar proteini ali pripete oznake specifično reagirajo s kolono za čiščenje. Tehnike so znane poznavalcem tega področja.Protein purification techniques include size separation chromatography, ion exchange chromatography, reverse phase chromatography, and affinity chromatography when proteins or attached labels specifically react with the purification column. The techniques are known to those skilled in the art.
Sestavljanje polipeptidnega materiala dosežemo ob ugodnih pogojih, ki omogočajo povezovanje elastinu podobnih segmentov preko segmentov za tvorbo ovitih vijačnic. Kadar je polipeptidni material sestavljen iz ene same vrste proteina, primerne pogoje najdemo s preizkušanjem topnosti in sekundarne strukture proteina topnega dela in še posebej precipitata v pufrih z različnim pH (ponavadi od pH 3 do pH 9), ionski jakosti, organskih topilih (npr. DMSO, acetonitril, trifloroetanol). Pomembna dejavnika pri samosestavljanju sta koncentracija fuzijskega proteina in temperatura. Polipeptidni material se tvori, ko je koncentracija proteina med 0.1 mg/mL in 20 mg/mL, ponavadi od 0.5 do 10 mg/mL. Samosestavljanje polipeptidnega materiala lahko sprožimo tudi s segrevanjem precipitata fuzijskega proteina preko najnižje temperature tališča, ki mu sledi počasno ohlajanje.The assembly of the polypeptide material is achieved under favorable conditions that allow the connection of elastin-like segments via segments to form wrapped helices. When the polypeptide material consists of a single type of protein, suitable conditions are found by testing the solubility and secondary structure of the protein of the soluble moiety and especially the precipitate in buffers of different pH (usually pH 3 to pH 9), ionic strength, organic solvents. DMSO, acetonitrile, trifluoroethanol). Fusion protein concentration and temperature are important factors in self-assembly. A polypeptide material is formed when the protein concentration is between 0.1 mg / mL and 20 mg / mL, usually 0.5 to 10 mg / mL. Self-assembly of the polypeptide material can also be triggered by heating the fusion protein precipitate above the lowest melting point, followed by slow cooling.
Kadar se polipeptidni material sestavi iz več različnih fuzijskih proteinov, se samosestavljanje ponavadi začne, ko zmešamo proteine v primernem molamem razmerju v pogojih ugodnih za samosestavljanje.When a polypeptide material is composed of several different fusion proteins, self-assembly usually begins when the proteins are mixed in a suitable molar ratio under conditions favorable to self-assembly.
Protein polipeptidnega materiala, ki vsebuje vsaj en elastinu podoben segment in vsaj dva segmenta za tvorbo ovitih vijačnic, lahko zmešamo v različnih razmerjih s proteinom, ki vsebuje tudi funkcionalno polipeptidno domeno. Glede na želene lastnosti polipeptidnega materiala, je lahko mešanica pripravljena iz različnih razmerij, pri čemer je protein, ki poleg vsaj enega elastinu podobnega segmenta in vsaj dveh segmentov za tvorbo ovitih vijačnic, vsebuje tudi funkcionalno polipeptidno domeno predstavlja manj kot 50% mešanice, bolj pogosto 20% in največkrat 1-5 % mešanice. Z mešanjem dveh proteinov, pri čemer segmenti za tvorbo ovitih vijačnic enega proteina tvorijo heterooligomere s segmenti za tvorbo ovite vijačnice drugega proteina, je mogoče regulirati začetek nastajanja polipeptidnega materiala. -20-A protein of a polypeptide material comprising at least one elastin-like segment and at least two segments to form coiled helices can be mixed in various proportions with a protein that also contains a functional polypeptide domain. Depending on the desired properties of the polypeptide material, the mixture may be prepared from different ratios, with a protein that contains less than 50% of the mixture in addition to at least one elastin-like segment and at least two segments for forming coiled helices, more often 20% and most often 1-5% of the mixture. By mixing the two proteins, the envelope-forming helix segments of one protein forming heterooligomers with the envelope-forming helix segments of the other protein, the onset of polypeptide material formation can be regulated. -20-
Protein, ki vsebuje funkcionalno polipeptidno domeno združeno s segmentom za tvorbo ovite vijačnice komplementarnim segmentu za tvorbo ovite vijačnice v polipeptidnem materialu, lahko prav tako dodamo polipeptidnemu materialu..A protein comprising a functional polypeptide domain combined with a helix-forming helix segment to complement the helix-helix segment in the polypeptide material may also be added to the polypeptide material.
Preko inkorporacije funkcionalnih delov, ki so združeni s segmenti za tvorbo ovite vijačnice, je mogoče polipeptidnemu materialu dodati funkcionalne lastnosti. Z dodajanjem peptidov, ki jih sestavljajo segmenti za tvorjenje ovite vijačnice, ki so v enem od dveh materialov, ki sestavljata polipeptidni material, je mogoče doseči razgradnjo polipeptidnega materiala, ki omogoča enostavno pridobivanje celic iz materiala.Through the incorporation of functional parts that are joined to segments to form a wrapped helix, functional properties can be added to the polypeptide material. By adding peptides composed of segments to form a coiled helix that are in one of the two materials that make up the polypeptide material, degradation of the polypeptide material can be achieved, allowing cells to be easily obtained from the material.
Uporaba polipeptidnega materialaUse of polypeptide material
Izum se nanaša tudi na možne uporabe polipeptidnega materiala. Osnova je, da se polipeptidni material lahko uporabi kot material/farmacevtski pripravek, ki spodbuja rast celic, tkiv ali organov, diferenciacijo celic, popravilo celic, reprogramiranje celic, citotoksično delovanje, za medicinski in farmacevtski material za živa človeška oz. živalska tkiva, za preprečevanje rasti patogenov.The invention also relates to possible uses of the polypeptide material. The basis is that the polypeptide material can be used as a material / pharmaceutical preparation that promotes cell, tissue or organ growth, cell differentiation, cell repair, cell reprogramming, cytotoxic action, for medical and pharmaceutical material for living human resp. animal tissues, to prevent the growth of pathogens.
Izraz "rast" ima splošen pomen in se nanaša na razvoj celic/tkiv/organov in delitev celic. Predstavljeni izum nudi okolje za gojenje katerega koli tipa celic, vključno z , vendar ne omejeno na: celice gladkih mišic, fibroblaste, keratinocite, epitelne, imunske in endotelne celice. Polipeptidni material posnema naravno okolje celic - ekstracelulami matriks, saj vključuje omrežje elstinu podobnih segmentov. Celice, tkiva in organi se lahko gojijo na površini polipeptidnega materiala, poleg tega pa se lahko celice razširijo v njegovo strukturo ali migrirajo vanj v primeru, da material tvori tridimentionalno strukturo.The term " growth " has a general meaning and refers to the development of cells / tissues / organs and cell division. The present invention provides an environment for the cultivation of any type of cell, including, but not limited to: smooth muscle cells, fibroblasts, keratinocytes, epithelial, immune, and endothelial cells. The polypeptide material mimics the natural environment of cells - the extracellular matrix, as it includes a network of elstin-like segments. Cells, tissues and organs can grow on the surface of a polypeptide material, and in addition, cells can expand into its structure or migrate into it in case the material forms a three-dimensional structure.
Celice, tkiva in organe je mogoče gojiti na polipeptidnemu materialu, ki zagotavlja pogoje ki podobne fiziološkim, na podoben način kot jih je mogoče gojiti v primerni posodi za celične kulture. Odvisno od hitrosti proliferacije celic, njihovega števila in gostote, ki so odvisni od tipa celic in namena uporabe,, je mogoče gojiti celice na polipeptidnem materialu ustrezno dolgo. V primeru gojenja celic na polipeptidnem materialu, ki dodatno vsebuje vsaj eno ali kombinacije večih, ponavadi 2 do 10, funkcionalnih polipeptidnih domen, ki spadajo med, vendar niso mejene na: rastne faktorje, molekule za adhezijo celic, molekule kemotakse, ligande receptorjev, protimikrobne peptide, faktorje za reprogramiranje, faktorje za -21- diferenciacijo celic, citotoksične ali citoststske polipeptide, kot so npr. epidermalni rastni faktor (EGF), fibroblastni rastni faktorji, nevronski rastni faktorji (NGF), še posebej SEQ ID NO: 24, protimikrobni peptidi kot so katelicideni in defenzini, predvsem katelicidin LL-37, še posebej SEQ ID NO: 16.Cells, tissues and organs can be grown on polypeptide material that provides conditions that are similar to physiological, in a similar way as they can be grown in a suitable container for cell cultures. Depending on the rate of cell proliferation, their number and density, which depend on the type of cells and the purpose of use, cells can be grown on polypeptide material for an appropriate length of time. In the case of culturing cells on a polypeptide material further comprising at least one or a combination of several, usually 2 to 10, functional polypeptide domains belonging to, but not limited to: growth factors, cell adhesion molecules, chemotaxis molecules, receptor ligands, antimicrobials peptides, reprogramming factors, cell-differentiation factors, cytotoxic or cytosthetic polypeptides such as e.g. epidermal growth factor (EGF), fibroblast growth factors, neuronal growth factors (NGF), in particular SEQ ID NO: 24, antimicrobial peptides such as cathelicidens and defensins, in particular cathelicidin LL-37, in particular SEQ ID NO: 16.
Funkcionalne polipepeptidne domene torej ni potrebno dodajati gojišču, saj je že vezana na polipeptidni material, na katerem rastejo celice, kar predstavlja veliko prednost. Kot je bilo že omenjeno, obstaja tudi možnost mešanja polipeptidnih materialov z različnimi funkcionalnimi polipeptidnimi domenami v poljubnih razmerjih, kar omogoča zagotavljanje zaželenih lastnosti za optimalno rast celic in nudi skoraj neskončno možnih kombinacij.It is therefore not necessary to add a functional polypeptide domain to the medium, as it is already bound to the polypeptide material on which the cells grow, which is a great advantage. As already mentioned, there is also the possibility of mixing polypeptide materials with different functional polypeptide domains in any ratio, which allows to provide the desired properties for optimal cell growth and offers almost infinitely possible combinations.
Dodatek peptidov, ki predstavljajo segment za tvorbo ovite vijačnice inkorporiran v polipeptidnem materialu, polipeptidnemu materialu lahko vodi do razpada materiala, kar predstavlja možnost za nežno odstranitev celic, ki jih lahko nato preštejemo, presadimo naprej ali uporabimo v nadalnji biokemijski analizi ali različnih tehnoloških aplikacijah. V nasprotju z znanimi metodami, ki vključujejo biokemične in fizikalne spremembe ali encimsko razgradnjo. Omenjeni pristop ne vpliva na ali poškoduje molekul na površini celic ali povzroča stres celicam.The addition of peptides, which represent a segment to form a coiled helix incorporated into the polypeptide material, can lead to the disintegration of the polypeptide material, providing the ability to gently remove cells that can then be counted, transplanted or used in further biochemical analysis or various technological applications. In contrast to known methods involving biochemical and physical changes or enzymatic degradation. This approach does not affect or damage molecules on the cell surface or cause stress to the cells.
Polipeptidni material je mogoče uporabiti kot material za medicinske in farmacentske namene za zdravljenje živega tkiva, predvsem človeškega in živalskega tkiva.The polypeptide material can be used as a material for medical and pharmaceutical purposes for the treatment of living tissue, especially human and animal tissue.
Polipeptidni material z ali brez celic ali tkiv, ki rastejo na površini, je mogoče uporabiti kot material za zdravljenje številnih poškodb ali bolezni tkiva in ga je mogoče vsaditi v telo s kirurškimi ali drugimi ustreznimi posegi.A polypeptide material with or without cells or tissues growing on the surface can be used as a material to treat a number of tissue injuries or diseases and can be implanted in the body by surgery or other appropriate procedures.
Izraz "zdravljenje" ima splošni pomen in se v opisu nanaša na postopek za odpravljanje napak okvarjenega ali poškodovanega človeškega ali živalskehga tkiva z uporabo polipeptidnega materiala.The term " treatment " has a general meaning and refers in the description to a procedure for correcting defects in damaged or damaged human or animal tissue using a polypeptide material.
Izraz "priprava medicinskega in farmacevtskega materiala" se nanaša na postopke, ki se jih poslužujemo, da uredimo polipeptidni material v tako obliko oziroma ga pripravimo v takem stanju, da se ga lahko uporabi kot npr. nadomestek za ranjeno tkivo, kot proteza ali kot farmacevtski material, kot so npr. ovoji za zdravljenje ran in opeklin.The term " preparation of medical and pharmaceutical materials " refers to the processes used to arrange the polypeptide material in such a form or to prepare it in such a state that it can be used as e.g. wound tissue replacement, as a prosthesis or as a pharmaceutical material, such as e.g. dressings for the treatment of wounds and burns.
Opisani polipeptidni material izzove minimalni ali nezaznavni imunski oz. vnetni odziv. V splošnem je v izumu opisani polipeptidni material mogoče uporabiti v vsaki situaciji, ki se tiče poškodb ali okvarjenega tkiva, ki je posledica operacije, travme, tumorja, degenerativne ali -22- ···· ··The described polypeptide material elicits minimal or undetectable immune or inflammatory response. 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 -22- ···· ··
druge bolezni oz. stanja. Polipeptidni material je mogoče uporabiti za popravilo naravnih elastičnih sistemov, še posebno tistih, v katerih sta naravno prisotna elastin in tropoelastin, z nadomeščanjem poškodovanega dela sistema, kot so npr. vezi, kite, stena krvne žile, ipd., s polipeptidnim materialom. Polipeptidni material je tudi mogoče operativno vnesti v človeka ali žival na mesto okvarjenega ali manjkajočega vaskulamega materiala in je tako uporaben kot vaskulami nadomestek oziroma za zapiranje poškodb. Polipeptidni material je uporaben za ponovno vzpostavitev strukturne in/ali funkcionalne integritete tkiva.other diseases or conditions. The polypeptide material can be used to repair natural elastic systems, especially those in which elastin and tropoelastin are naturally present, by replacing the damaged part of the system, such as e.g. ligaments, tendons, blood vessel wall, etc., with polypeptide material. The polypeptide material can also be surgically inserted into a human or animal at the site of defective or missing vascular material and is as useful as a vascular replacement or to close injuries. The polypeptide material is useful for restoring the structural and / or functional integrity of the tissue.
Celice, ki so se namnožile/diferencirale na polipeptidnem materialu je možno odstraniti iz strukture in jih naprej gojiti in vitro v posodi za celične kulture. Kasneje jih lahko posredujemo osebku in jih uporabimo kot nadomestilo tkiva ali organa.Cells that have multiplied / differentiated on the polypeptide material can be removed from the structure and further cultured in vitro in a cell culture dish. They can later be passed on to a specimen and used as a tissue or organ replacement.
Uporaba polipeptidnega materiala, ki vsebuje tudi funkcionalno polipeptidno domeno, npr. rastne faktorje, za zdravljenje živega človeškega ali živalskega ima določene prednosti pred zdravljenjem z uporabo rastnih faktorjev v topni obliki. Topni rastni faktorji imajo lahko nekatere nezaželene učinke, še posebno in vivo kot npr. stimulacija rasti celic, ki tekmujejo s tarčnimi celicami, zaradi česar te celice prerastejo tarčne celice, ali difuzija v krvni obtok in izražanje njihovih efektov na drugih mestih.. Z dodatkom funkcionalne polipeptidne domene polipeptidnemu materialu dosežemo lokalni vpliv funkcionalne domene na tarčne celice. Opisani material tudi ponuja možni način za inhibicijo rasti nezaželenega tkiva z lokalnim citotoksičnim ali citostatskim učinkom na mestu aplikacije, predvsem, vendat ne omejeno na rakasto tkivo ipd.The use of a polypeptide material that also contains a functional polypeptide domain, e.g. growth factors, for the treatment of live human or animal has certain advantages over treatment using growth factors in soluble form. Soluble growth factors can have some side effects, especially in vivo such as e.g. stimulating the growth of cells competing with target cells, causing these cells to outgrow target cells, or diffusing them into the bloodstream and expressing their effects elsewhere. By adding a functional polypeptide domain to the polypeptide material, local influence of the functional domain on target cells is achieved. The described material also offers a possible way to inhibit the growth of unwanted tissue with a local cytotoxic or cytostatic effect at the site of application, in particular, vendat not limited to cancerous tissue and the like.
Kadar so epitelne celice nasajene z namenom rekonstrukcije dela epiderma, polipeptidni material lahko služi kot osnova kože za transplantacijo na prejemnika. Polipeptidni material je uporaben tudi za konstrukcijo protez, ki vsebujejo elastin podoben človeškemu, npr. cevke za nadomestilo krvnih žil in ovoji za zdravljenje ran in opeklin. Proteza lahko postane trajni nadomestek za tkivo, saj pacientove celice, vključno z endotelnimi, infiltrirajo polipeptidni material. Polipeptidni material je mogoče uporabiti tudi za prekrivanje površin kakršnega koli tipa protez, tudi protez iz sintetičnih materialov in/ali kovin.When epithelial cells are implanted for the purpose of reconstructing part of the epidermis, the polypeptide material can serve as the basis of the skin for transplantation to the recipient. The polypeptide material is also useful for the construction of prostheses containing human-like elastin, e.g. blood vessel replacement tubes and dressings for the treatment of wounds and burns. The prosthesis can become a permanent tissue replacement as patient cells, including endothelial cells, infiltrate the polypeptide material. The polypeptide material can also be used to cover the surfaces of any type of prosthesis, including prostheses made of synthetic materials and / or metals.
Izraz "proteza" se nanaša na vsak material vsajen v telo, vključno z nadomestki krvnih žil, srčnih zaklopk ali zaplate materiala. Uporabi se lahko tudi kot ovoj, ki spodbuja zdravljenje ran in opeklin.The term " prosthesis " refers to any material implanted in the body, including blood vessel replacements, heart valves, or patches of material. It can also be used as a wrap that promotes healing of wounds and burns.
Predlagatelji odkrili tudi, da polipeptidni material, kadar poleg vsaj enega elastinu podobnega segmenta in vsaj dveh segmentov za tvorbo ovitih vijačnic vsebuje tudi funkcionalno -23- polipeptidno domeno, ki jo predstavlja protimikrobni peptid, kot so katelicidini in defenzini, predvsem katelicidin LL-37, še posebej SEQ ID NO: 16., ali domena za vezavo kovin, še posebej SEQ ID NO: 26, ki tvori srebrne nanodelce, uporaben tudi za preprečevanje rasti patogenov.. Prednost protimikrobnih peptidov in kovin, predvsem srebra, je v njihovem širokem spektru protimikrobnega delovanja.Proponents have also found that a polypeptide material, when in addition to at least one elastin-like segment and at least two segments for the formation of enveloped helices, also contains a functional -23- polypeptide domain represented by an antimicrobial peptide such as cathelicidins and defensins, especially cathelicidin LL-37, in particular SEQ ID NO: 16., or a metal binding domain, in particular SEQ ID NO: 26, which forms silver nanoparticles, also useful for preventing the growth of pathogens. The advantage of antimicrobial peptides and metals, especially silver, is in their wide range antimicrobial action.
Izraz "patogeni" v tem opisu se nanaša na pogoste bakterije, glive, kvasovke in viruse, ki povzročajo bolezni. Predvsem se nanaša na pogoste bakterijske povzročitelje bolezni kot so, vendar niso omejeni na, gram pozitivne bakterije S. aureus, S. pyogenes, S. pneumoniae, in gram negativne bakterije H. influenzae, K. pneumoniae, L. pneumophila, P. aeruginosa, E. coli.The term " pathogens " in this description it refers to common bacteria, fungi, yeasts and viruses that cause diseases. In particular, it refers to common bacterial pathogens such as, but not limited to, gram-positive bacteria S. aureus, S. pyogenes, S. pneumoniae, and gram-negative bacteria H. influenzae, K. pneumoniae, L. pneumophila, P. aeruginosa , E. coli.
Infekcije se pogosto pojavijo kot posledica katerega koli posega v tkivo. Glede na to, da je polipeptidni material mogoče uporabiti kot nadomedtilo za okvarjeno ali poškodovano naravno elastično tkivo in ga je možno presaditi v prejemnika, predstavlja določeno prednost, če polipeptidni material tudi preprečuje rast patogenov in in tako ublaži ali prepreči možno okužbo. Poleg tega je polipeptidni material uporaben tudi za konstrukcijo protez, npr. cevk za nadomeščanje krvnih žil ali ovojev za druge uporabe kot je zdravljenje ran in opeklin kot je bilo že omenjeno ali za prektivanje površin protez. Proteze pogosto predstavljajo grožnjo bolnikom zaradi razvoja biofilmov, kar lahko vodi do mnogih življensko nevarnih okužb, vključno z okužbami ki jih povzročajo npr. P. aeruginosa in S. aureus, dve bakteriji, ki jih pogosto najdemo v biofilmih in tudi pri infekcijah opeklin. V tem kontekstu opisana iznajdba predstavlja možnost za lajšanje in preprečevanje okužb, ki jih povzročajo patogeni prisotni na protezah in opeklinah.Infections often occur as a result of any intervention in the tissue. Given that a polypeptide material can be used as a substitute for damaged or damaged natural elastic tissue and can be transplanted into a recipient, it has the advantage that the polypeptide material also prevents the growth of pathogens and thus alleviates or prevents possible infection. In addition, the polypeptide material is also useful for the construction of prostheses, e.g. tubes for replacing blood vessels or sheaths for other uses such as the treatment of wounds and burns as already mentioned or for trimming the surfaces of dentures. Prostheses often pose a threat to patients due to the development of biofilms, which can lead to many life-threatening infections, including infections caused by e.g. P. aeruginosa and S. aureus, two bacteria often found in biofilms and also in burn infections. In this context, the present invention provides an opportunity to alleviate and prevent infections caused by pathogens present on dentures and burns.
Glede na to, daje srebro neselektivno in izjemno aktivno v majhnih količinah, so predlagatelji odkrili, da polipeptidni material, ki vsebuje domeno za vezavo kovin, predvsem SEQ ID NO: 26, tvori srebrne nanodelce in predstavlja še posebej obetajoč material za preprečevanje rasti patogenov, še posebej uporaben za povijanje ran in za proteze.Given that silver is nonselective and extremely active in small amounts, the proponents found that a polypeptide material containing a metal binding domain, in particular SEQ ID NO: 26, forms silver nanoparticles and is a particularly promising material for preventing the growth of pathogens, especially useful for wound dressings and prostheses.
Spodaj so navedeni primeri postopkov, načrtovani z namenom ilustracije iznajdbe. Opisi primerov nimajo namena omejevanja iznajdbe in bi morali biti razumljeni kot demonstracija iznajdbe. -24- • · « · • · • ·The following are examples of processes designed to illustrate the invention. The case descriptions are not intended to limit the invention and should be construed as a demonstration of the invention. -24- • · «· • · • ·
Primeri izvedbeExamples of implementation
Primer 1: Priprava DNK konstruktaExample 1: Preparation of a DNA construct
Tukaj opisani polipeptidni material je bil načrtovan tako, da združuje segmente podobne elastinu, segmente za tvorbo ovitih vijačnic ter funkcionalne domene. Aminokislinska zaporedja segmentov za tvorbo heterodimemih paralelnih ovitih vijačnic (SEQ ID: 10, SEQ ID: 12, SEQ ID: 28, SEQ ID: 30; SEQ ID:32, SEQ ID: 34, SEQ ID: 36, SEQ ID: 38) so bila načrtovana na podlagi dejstev o sestavljanju segmentov za tvorbo ovitih vijačnic, ki so znana izvedencem na tem področju, z namenom povečanja specifičnosti interakcij med segmenti za tvorbo ovitih vijačnic. Aminokislinsko zaporedje segmentov za tvorbo homodimerne paralelne ovite vijačnice, je bilo vzeto iz GCN4 peptida (SEQ ID: 14). Trimerizacijsko zaporedje za tvorbo ovite vijačnice AEA (SEQ ID: 26), ki tvori srebrne nanodelce, je bilo načrtovalno na podlagi dejstev o sestavljanju segmentov za tvorbo ovitih vijačnic, ki so znana izvedencem na tem področju, s ponavljanjem treh segmentov, ki se združijo v stopničasti heterotrimer in imajo na mestih b, c in / heptade ovite vijačnice aminokislinske ostaneke Ala (b), Ala (c) and Glu (/)The polypeptide material described herein has been designed to combine elastin-like segments, helix-forming segments, and functional domains. Amino acid sequences of segments for the formation of heterodyme parallel helixes (SEQ ID: 10, SEQ ID: 12, SEQ ID: 28, SEQ ID: 30; SEQ ID: 32, SEQ ID: 34, SEQ ID: 36, SEQ ID: 38) were designed based on the facts about the assembly of segments for the formation of wrapped helices, known to experts in this field, in order to increase the specificity of the interactions between the segments for the formation of wrapped helices. The amino acid sequence of the segments to form a homodimeric parallel envelope helix was taken from the GCN4 peptide (SEQ ID: 14). The trimization sequence for the formation of the AEA coiled helix (SEQ ID: 26), which forms silver nanoparticles, was designed based on the facts about the assembly of segments for the formation of the coiled helix known to those skilled in the art, by repeating the three segments stepped heterotrimer and have amino acid residues Ala (b), Ala (c) and Glu (/) at the b, c and / heptade-wound helices.
Elastinu podobni segmenti so bili izbrani izmed ponovitev pentapeptida (SEQ ID: 18).Elastin-like segments were selected from pentapeptide repeats (SEQ ID: 18).
Funkcionalne domene, ki so bile izbrane za primer so: nevronski rastni dejavnik (SEQ ID: 24), protimikrobni peptid LL-37 (SEQ ID: 16). Izbrane so bile izmed naravnih zaporedij glodalskih in človeških proteinov.The functional domains selected for the example are: neuronal growth factor (SEQ ID: 24), antimicrobial peptide LL-37 (SEQ ID: 16). They were selected from natural sequences of rodent and human proteins.
Fuzijski proteini lahko vključujejo signalno zaporedje za lokalizacijo proteina in peptidno oznako za detekcijo ali čiščenje proteina. DNK zaporedja optimizirana za ekspresijo proteina v želenem gostitelju (E.coli) so bila načrtovana na podlagi aminokislinskih zaporedij in s pomočjo programa Gene Designer DNA2.0 Inc. verzija 1.0.0.1 (DNA2.0 Headquarter 1430 0'Brien Drive, Suite E, Menlo Park, CA 94025, USA). DNK zaporedja so bila naročena pri MR.Gene GmbH (Im Gewerbepark B32, D-93059 Regensburg) in izrezani z restrikcijskimi endonukleazami. LL-37 DNK zaporedje je bilo vzeto iz plazmida z odprtim bralnim okvirjem klona katelicidin protimikrobnega peptida vrste Homo sapiens (ČAMP). Del gena, ki kodira LL-37, je bil pomnožen s PCR. DNK konstrukti in pripadajoči fuzijski proteini so opisani v Tabeli 1. Podrobni opisi in funkcije posameznih DNK in/ali proteinskih produktov so v Tabeli 2. Vsi DNK konstrukti -25- « m imajo start kodon (ATG) pred histidinsko oznako. Konstrukti so bili klonirani v pET-31b(+) vektor za visoko stopnjo ekspresije peptidne sekvence priključene na protein ketosteroid izomerazo. Ekspresijska kaseta vsebuje v smeri 5' proti 3' T7 promotor, kodirajoče zaporedje za KSI in multiplo mesto za kloniranje za fuzijski protein ter T7 terminator. Ti regulatomi elementi omogočajo izražanje proteina v prokariontski celični liniji E.coli, ki izraža T7 RNA polimerazo. DNK konstrukti so bili pripravljeni z uporabo metod molekularne biologije, ki so opisane v priročniku molekularne biologije (Sambrook J., Fritsch E.F., Maniatis T. 1989. Molecular cloning: A laboratory manual. 2nd ed. New York, Cold Spring Harbor Laboratory Press: 1659 str.). Plazmidi, konstrukti in vmesni konstrukti so bili vneseni s kemično transformacijo v bakterije E. coli DH5a ali BL21 (DE3) pLysS.Fusion proteins may include a signal sequence for localizing the protein and a peptide label for detecting or purifying the protein. DNA sequences optimized for protein expression in the desired host (E. coli) were designed based on amino acid sequences and using Gene Designer DNA2.0 Inc. version 1.0.0.1 (DNA2.0 Headquarter 1430 0'Brien Drive, Suite E, Menlo Park, CA 94025, USA). DNA sequences were ordered from MR.Gene GmbH (Im Gewerbepark B32, D-93059 Regensburg) and excised with restriction endonucleases. The LL-37 DNA sequence was taken from an open-reading plasmid clone of the Homo sapiens antimicrobial peptide (CHAMP) clonicidin clone. The portion of the gene encoding LL-37 was amplified 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 given in Table 2. All DNA constructs -25- «m have a start codon (ATG) in front of the histidine tag. The constructs were cloned into a pET-31b (+) vector for a high degree of expression of the peptide sequence attached to the protein ketosteroid isomerase. The expression cassette contains a 5 'to 3' T7 promoter, a coding sequence for KSI and a multiple cloning site for the fusion protein, and a T7 terminator. These regulatory elements allow the expression of a protein in the prokaryotic E.coli cell line that expresses T7 RNA polymerase. DNA constructs were prepared using the molecular biology methods described in the Molecular Biology Handbook (Sambrook J., Fritsch EF, Maniatis T. 1989. Molecular cloning: A laboratory manual. 2nd ed. New York, Cold Spring Harbor Laboratory Press: 1659 p.). Plasmids, constructs, and intermediates were introduced by chemical transformation into E. coli DH5a or BL21 (DE3) pLysS.
Fuzijski proteini, ki pripadajo načrtovanim DNK konstruktom so v splošnem sestavljeni iz vsaj enega elastinu podobnega segmenta in vsaj dveh segmentov za tvorbo ovitih vijačnic.„poleg tega pa izbirno vsebujejo tudi funkcionalno polipeptidno domeno (npr. NGF in/ali LL-37). KSI-DP domena omogoča ekspresijo proteinov v inkluzij skih telesih in kislo cepitev aspartat-prolin (DP) vezi.Fusion proteins belonging to the engineered DNA constructs generally consist of at least one elastin-like segment and at least two envelope-forming helix segments. 'In addition, they optionally contain a functional polypeptide domain (eg NGF and / or LL-37). The KSI-DP domain allows the expression of proteins in inclusion bodies and the acid cleavage of the aspartate-proline (DP) bond.
Tabela 1: Fuzijski proteini uporabljeni za demonstracijo iznajdbeTable 1: Fusion proteins used to demonstrate the invention
Nukleotidno/ami Št. Ime Sestava konstrukta nokislinsko zaporedje plazmid 1 KSI-DP-Histag-LL3 7 -ELST-GCN-ELST DP-Hista-LL37-ELST-GCN-ELST -T7t SEQ ID NO: 1/ SEQ ID NO: 2 pET-31b(+) 2 KSI-DP-Histag-LL3 7 -ELST-GCN-P2-ELST -NGF DP-Hista-LL37-ELST-GCN-P2-ELST-NGF -T7t SEQ ID NO: 3/ SEQ ID NO: 4 pET-31b(+) 3 Pl Sintetični SEQ ID NO: 9/ SEQ ID NO: 10 4 P2 Sintetični SEQ ID NO: 11/ SEQ ID NO: 12 5 KSI-DP-Histag-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-Hista-LL3 7-ELST-GCN-P2-ELST -T7t SEQ ID NO: 7/ SEQ ID NO: 8 pET-31b(+) 7 KSI-DP-Histag-ELST-GCN-P2-ELST - AEA DP-His,a-ELST-GCN-P2- ELST-AEA-T7t SEQ ID NO: 19/ SEQ ID NO: 20 pET-31b(+) -26-Nucleotide / s No. Name Composition composition acid sequence plasmid 1 KSI-DP-Histag-LL3 7 -ELST-GCN-ELST DP-Hista-LL37-ELST-GCN-ELST -T7t SEQ ID NO: 1 / SEQ ID NO: 2 pET-31b (+ ) 2 KSI-DP-Histag-LL3 7 -ELST-GCN-P2-ELST -NGF DP-Hista-LL37-ELST-GCN-P2-ELST-NGF -T7t SEQ ID NO: 3 / SEQ ID NO: 4 pET- 31b (+) 3 Pl Synthetic SEQ ID NO: 9 / SEQ ID NO: 10 4 P2 Synthetic SEQ ID NO: 11 / SEQ ID NO: 12 5 KSI-DP-Histag-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-Hista -LL3 7-ELST-GCN-P2-ELST -T7t SEQ ID NO: 7 / SEQ ID NO: 8 pET-31b (+) 7 KSI-DP-Histag-ELST-GCN-P2-ELST - AEA DP-His, a-ELST-GCN-P2- ELST-AEA-T7t SEQ ID NO: 19 / SEQ ID NO: 20 pET-31b (+) -26-
Tabela 2: Seznam genov, funkcij in število iz baze podatkov ter aminokislinsko/nukleotidno zaporedje, ki predstavlja meje uporabljenih delov genov.Table 2: List of genes, functions and number from the database and amino acid / nucleotide sequence representing the boundaries of the gene parts used.
Ime gena Swiss Prot No. Nukleotidno zaporedje funkcija function T7p promoter T7t terminator Histag HHHHHH aminokislinski označevalec KSI P00947 SEQ ID NO: 21 AK:1-125 (SEQ ID NO: 22) ketosteroid isomeraza DP - GATCCT DP dipeptid aspartat-prolin Pl - SEQ ID NO: 9 SEQ ID NO: 10 segment za tvorbo ovite vijačnice, ki tvori heterodimer s P2 P2 - SEQ ID NO: 11 SEQ ID NO: 12 segment za tvorbo ovite vijačnice, ki tvori heterodimer s Pl GCN _ SEQ ID NO: 13 SEQ ID NO: 14 segment za tvorbo homodimerne ovite vijačnice LL37 P49913 SEQ ID NO: 15 AK: 134-170 SEQ ID NO: 16 protimikrobni peptid ELST SEQ ID NO: 17 SEQ ID NO: 18 segment podoben elastinu NGF P01139 SEQ ID NO: 23 AK: 19-241 (SEQ ID NO: 24) beta-nevronski rastni faktor (Beta-NGF) -mišji AEA - SEQ ID NO: 25 SEQ ID NO: 26 domena za tvorjenje srebrnih nanodelcev P3 - SEQ ID NO: 27 SEQ ID NO: 28 segment za tvorbo ovite vijačnice, ki tvori heterodimer s P4 P4 - SEQ ID NO: 29 SEQ ID NO: 30 segment za tvorbo ovite vijačnice, ki tvori heterodimer s P3 P5 - SEQ ID NO: 31 SEQ ID NO: 32 segment za tvorbo ovite vijačnice, ki tvori heterodimer s P6 P6 - SEQ ID NO: 33 SEQ ID NO: 34 segment za tvorbo ovite vijačnice, ki tvori heterodimer s P5 P7 - SEQ ID NO: 35 SEQ ID NO: 36 segment za tvorbo ovite vijačnice, ki tvori heterodimer s P 8 P8 - SEQ ID NO: 37 SEQ ID NO: 38 segment za tvorbo ovite vijačnice, ki tvori heterodimer s P7 -27-Gene name Swiss Prot No. Nucleotide sequence function 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 Pl - SEQ ID NO: 9 NO: 10 segment for forming a wrapped helix forming a heterodimer with P2 P2 - SEQ ID NO: 11 SEQ ID NO: 12 segment for forming a wrapped helix forming a heterodimer with Pl GCN _ SEQ ID NO: 13 SEQ ID NO: 14 segment for forming homodimeric coiled helix LL37 P49913 SEQ ID NO: 15 AK: 134-170 SEQ ID NO: 16 antimicrobial peptide ELST SEQ ID NO: 17 SEQ ID NO: 18 elastin-like segment NGF P01139 SEQ ID NO: 23 AK: 19-241 (SEQ ID NO: 24) beta-neuronal growth factor (Beta-NGF) -mouse AEA - SEQ ID NO: 25 SEQ ID NO: 26 domain for the formation of silver nanoparticles P3 - SEQ ID NO: 27 SEQ ID NO: 28 segment for for forming a helix forming a heterodimer with P4 P4 - SEQ ID NO: 29 SEQ ID NO: 30 segment for forming a helix forming a heterodimer er s P3 P5 - SEQ ID NO: 31 SEQ ID NO: 32 segment for forming a helix forming a heterodimer with P6 P6 - SEQ ID NO: 33 SEQ ID NO: 34 segment for forming a helix forming a heterodimer with P5 P7 - SEQ ID NO: 35 SEQ ID NO: 36 segment for forming a wrapped helix forming a heterodimer with P 8 P8 - SEQ ID NO: 37 SEQ ID NO: 38 segment for forming a wrapped helix forming a heterodimer with P7 -27-
Primer 2: Produkcija fuzijskega proteina sestavljenega it elastinu podobnega segmenta in segmentov za tvotbo ovitih vijačnic, protimikrobnim peptidom in NGFExample 2: Production of a fusion protein composed of an elastin-like segment and segments for the formation of coiled helices, antimicrobial peptides and NGF
Pripravljenih je bilo več konstruktov, da bi dokazali možnost produkcije fuzijskih proteinov, ki so našteti v Tabeli 1 (SEQ ID No.l, 2, 5, 6).Several constructs were prepared to demonstrate the possibility of producing the fusion proteins listed in Table 1 (SEQ ID No.l, 2, 5, 6).
Plazmidi z zapisom (v odprtem bralnem okvirju) za fuzijske proteine, ki so našteti v Tabeli 2, so bili s kemijsko transformacijo vnešeni v kompetentne E. coli BL21 (DE3) pLysS celice. Izbrane bakterijske kolonije, ki so zrasle na LB ploščah z izbranim antibiotikim (ampicilin), smo precepil v 10 ml tekočega LB gojišča z dodanim izbranim antibiotikom. Po nekaj urah rasti na 37°C smo 10-100 pL kulture precepili v 100 mL izbranega rastnega medija, ki se je nato stresal čez noč na 37°C. Naslednji dan smo prekonočno kulturo 20- do 50-krat razredčili, s čimer je bila dosežena OD600 razredčene kulture med 0.1 in 0.2. Gojitvene posode s 500 ml razredčene kulture so bile nameščene na stresalnik, bakterije pa so bile gojene dokler ni OD600 dosegla 0.6-0.8. Ekspresijo proteina smo inducirali z dodatkom induktorja IPTG (0.4 mM ali 1 mM). Štiri ure po indukciji smo brozgo centrifugirali, bakterijske celice smo resuspendirali v pufru za lizo (Tris pH 8.0, 0.1% deoksiholata z dodano mešanico proteaznih inhibitorjev) in zmrznili na -80°C vsaj čez noč. Odtajano suspenzijo celic smo nadalje lizirali s sonikacijo. ki ji je sledilo centrifugiranje. Oborina (celične membrane, inkluzijska telesa) in supematant smo za potrditev ekspresije konstruktov preverili z SDS-PAGE in Westem blot analizo z uporabo anti-His-tag protiteles kot primarnih protiteles, kadar je bilo potrebno. Načrtovani fuzijski proteini so bili večinoma prisotni v netopnem delu (inkluzijska telesa), ki so bila sestavljena iz >80% želenega proteina. To je bila posledica fuzije s KSI domeno na N-terminalnem (končnem???) delu proteina. Inkluzijska telesa smo dvakrat sprali s pufrom za lizo, dvakrat z 2M ureo v 10 mM Tris pH 8.0 in enkrat z MiliQ vodo. Rezultat je bil ponavadi >95% čistoča proteina. V primeru, da je oborina še vedno vsebovala nečistoče, smo inkluzijska telesa raztopili v 6 M GdnHCl, pH 8.0 in nanesli na Ni2+-NTA kolono. Čiščenju pod denaturirajočimi pogoji je potekalo po navodilih izdelovalca. Po eluciji z 250 mM imidazolom v 100 mM Na3P04, 10 mM Tris pH 5.8, smo frakcije, ki so vsebovale protein združili in dvakrat dializirali proti 10 mM Hepes pH 7.5 ali MilliQ vodi. -28-The transcribed plasmids (in the open reading frame) for the fusion proteins listed in Table 2 were introduced into the competent E. coli BL21 (DE3) pLysS cells by chemical transformation. Selected bacterial colonies grown on LB plates with the selected antibiotic (ampicillin) were inoculated into 10 ml of liquid LB medium with added selected antibiotic. After a few hours of growth at 37 ° C, 10-100 pL of culture was inoculated into 100 mL of the selected growth medium, which was then shaken overnight at 37 ° C. The next day, the overnight culture was diluted 20 to 50 times to achieve an OD600 diluted culture between 0.1 and 0.2. Cultivation vessels with 500 mL of diluted culture were placed on a shaker and bacteria were grown until OD600 reached 0.6–0.8. Protein expression was induced by the addition of an IPTG inducer (0.4 mM or 1 mM). Four hours after induction, the slurry was centrifuged, the bacterial cells were resuspended in lysis buffer (Tris pH 8.0, 0.1% deoxycholate with added protease inhibitor mixture) and frozen at -80 ° C at least overnight. The thawed cell suspension was further lysed by sonication. followed by centrifugation. Precipitation (cell membranes, inclusion bodies) and supernatant were verified by SDS-PAGE and Western blot analysis to confirm the expression of the constructs using anti-His-tag antibodies as primary antibodies when necessary. The planned fusion proteins were mostly present in the insoluble part (inclusion bodies), which consisted of > 80% of the desired protein. This was due to fusion with the KSI domain on the N-terminal (terminal ???) portion of the protein. Inclusion bodies were washed twice with lysis buffer, twice with 2M urea in 10 mM Tris pH 8.0 and once with MiliQ water. The result was usually > 95% protein purity. In case the precipitate still contained impurities, the inclusion bodies were dissolved in 6 M GdnHCl, pH 8.0 and applied to a Ni2 + -NTA column. Cleaning under denaturing conditions was performed according to the manufacturer's instructions. After elution with 250 mM imidazole in 100 mM Na3PO4, 10 mM Tris pH 5.8, the protein-containing fractions were combined and dialyzed twice against 10 mM Hepes pH 7.5 or MilliQ water. -28-
Kadar je bil protein prisoten v supematantu, smo supematant nanesli na Ni2+-NTA kolono in očistili pri nativnih pogojih. Po eluciji z 250 mM imidazolom pH 5.8 ali 500 mM imidazolom pH 8.0, smo frakcije, ki so vsebovale protein, združili in dvakrat dializirali proti 10 mM Hepes pH 7.5 ali drugemu ustreznemu pufru.When the protein was present in the supernatant, the supernatant was applied to a Ni2 + -NTA column and purified under native conditions. After elution with 250 mM imidazole pH 5.8 or 500 mM imidazole pH 8.0, the protein-containing fractions were pooled and dialyzed twice against 10 mM Hepes pH 7.5 or other appropriate buffer.
Primer 3: Priprava materialaExample 3: Preparation of material
Pri ustreznih pogojih je protein z elastinu podobnimi segmenti in segmenti, za tvorbo ovitih vijačnic (paralelne heterodimeme ali homodimeme), nagnjen k tvorbi polipeptidnega materiala, ki se ga lahko uporabi za rast evkarintskih celic. Segmenti za tvorbo ovitih vijačnic, oligomerizirajo z drugimi, kar ima za posledico nastanek zamreženega materiala.Under appropriate conditions, a protein with elastin-like segments and segments, for the formation of enveloped helices (parallel heterodimes or homodimes), is prone to the formation of polypeptide material that can be used to grow eukaryotic cells. The segments for the formation of wrapped helices, oligomerize with others, resulting in the formation of crosslinked material.
Pri iskanju pogojev, v katerih so fuzijski proteini topni, smo protein denaturiranan v 6M gvanidinijevem HC1 približno 100-kratno redčili,, s pufri z različnimi pH vrednostmi (citratno acetatni pufer pH 2 in pH 3, acetatni pufri pH 4, pH 5, fosfatni pufri pH 5, pH 6, pH 7, pufer Hepes pH 7.5, pufer Tris pH 8, karbonatni pufer pH 9, pH 10), različno ionsko jakostjo (100 mM, 300 mM, 1 M, 2M NaCl) in v različnih organskih topilih z do 20% acetonitrila, DMSO, metanola ali do 50% trifluoroetanola. Izmerili smo absorbcijski spekter proteina. V primeru tipičnih proteinskih spektrov smo vzorca analizirali s CD spektroskopijo z namenom določitve sekundarne strukture proteina in temperaturne stabilnosti. Ko so bili določeni primerni pogoji, smo denaturirane proteine dializirali proti izbranim pufrom.In search of conditions under which fusion proteins are soluble, the protein denatured in 6M guanidinium HCl was diluted approximately 100-fold with buffers of different pH values (citrate acetate buffer pH 2 and pH 3, acetate buffers pH 4, pH 5, phosphate buffers pH 5, pH 6, pH 7, Hepes buffer pH 7.5, Tris buffer pH 8, carbonate buffer pH 9, pH 10), different ionic strength (100 mM, 300 mM, 1 M, 2M NaCl) and in different organic solvents with up to 20% acetonitrile, DMSO, methanol or up to 50% trifluoroethanol. The protein absorption spectrum was measured. In the case of typical protein spectra, the samples were analyzed by CD spectroscopy in order to determine the secondary structure of the protein and temperature stability. Once appropriate conditions were determined, denatured proteins were dialyzed against selected buffers.
Primer 4: Protimikrobno delovanje materiala, ki vsebuje protimikrobne peptide Protimikrobno delovanje materiala, ki vsebuje protimikrobne peptide, smo preverili z antibiogramom. Občutljivost bakterije E. coli DH5a na protimikrobni peptid je bila preverjena s semi-kvantitivno metodo, ki temelji na difuziji (Kirby-Bauer metoda). 20 pL prekonočne kulture E. coli DH5a smo premazali čez ploščo agarja s premerom 10 cm (petrijevka), da smo dosegli konfluentno rast. Nato smo na plošče položili 6 majhnih diskov in jih impregnirali z različnimi koncentracijami materiala z protimikrobnim peptidom (LL-37) od 0,1-10 mg/ml proteina in MiliQ, ki je služila kot negativna kontrola. Po 16 h inkubacije na 37°C je bila plošča pregledana za cone zbistritve. Protimikrobno delovanje je bilo odvisno od koncentracije materiala (Slika 2). -29-Example 4: Antimicrobial activity of a material containing antimicrobial peptides The antimicrobial activity of a material containing antimicrobial peptides was checked with an antibiogram. The susceptibility of E. coli DH5a to the antimicrobial peptide was verified by a semi-quantitative diffusion-based method (Kirby-Bauer method). 20 μL of overnight culture of E. coli DH5a was coated over a 10 cm diameter agar plate (petri dish) to achieve confluent growth. We then placed 6 small discs on the plates and impregnated them with different concentrations of material with antimicrobial peptide (LL-37) of 0.1–10 mg / ml protein and MiliQ, which served as a negative control. After 16 h of incubation at 37 ° C, the plate was inspected for clarification zones. The antimicrobial activity was dependent on the material concentration (Figure 2). -29-
Primer 5: Diferenciacija živčnih celic na polipeptidnem materialu sestavljenem in dveh elastinu podobnih segmentov, dveh segmentov za tvorbo ovitih vijačnic in NGFExample 5: Differentiation of nerve cells on a polypeptide material composed of two elastin-like segments, two segments for the formation of coiled helices and NGF
Linija celic podganjega feokromocitoma PC 12 je uveljavljen model za formacijo nevritov inducirano s nevronskim rastnim faktorjem (NGF). Dokazano je bilo, da dodatek gangliozidov mediju PC 12 celic pospeši za formacijo nevritov inducirano z nevronskim rastnim faktorjem (NGF). Nevronski rastni faktor pomaga pri preživetju in diferenciaciji senzornih in simpatičnih nevronov. Vpliva na TrkA in receptor za nevronski rastni faktor z nizko afiniteto (ang. Low-Affmity Nerve Grovvth Factor Receptor - LNGFR).The PC 12 rat pheochromocytoma cell line is an established model for neural growth factor-induced neurite formation (NGF). The addition of gangliosides to PC 12 cell medium has been shown to accelerate neuronal growth factor-induced neurite formation (NGF). Neuronal growth factor helps in the survival and differentiation of sensory and sympathetic neurons. It affects TrkA and the Low-Affmity Nerve Growth Factor Receptor (LNGFR).
Poskus smo izvedli v mikroskopirnih komoricah z 8 luknjami (Ibidi). Po nastanku polipeptidnega materiala smo v luknje nacepili celilce PC 12 v koncentraciji 0,25 χ 105 celic na luknjo. Dodali smo rastni medij F12K (Gibco) dopolnjen z 2,5% temperaturno inaktiviranega fetalnega govejega seruma in 15% temperaturno inaktiviranega konjskega seruma. Slike so bile posnete po 48h inkubacije na 37°C s 5% CO2 v atmosferi. Nastanek neuritov je prikazan na sliki 3D. NGF v polipeptidnem materialu ne povzroči morfoloških sprememb na HEK293 celicah (človeške embrionalne ledvične celice 293 - ang. Human Embryonic Kidney 293 celiš) (Slika 3B). Žive celice smo pregledovali na konfokalnem mikroskopu Leica TCS SP5 na Leica DMI 6000 CS stojalu. Uporabljali smo 63x oljni imerzijski objektiv.. Slike smo pridobili s programom LAS AF 1.8.0. Leica Microsystems.The experiment was performed in 8-hole microscopy chambers (Ibidi). After the formation of the polypeptide material, PC 12 cells were inoculated into the wells at a concentration of 0.25 χ 105 cells per well. F12K (Gibco) growth medium supplemented with 2.5% temperature-inactivated fetal bovine serum and 15% temperature-inactivated horse serum was added. Images were taken after 48 h of incubation at 37 ° C with 5% CO2 in the atmosphere. The formation of neurites is shown in a 3D image. NGF in the polypeptide material does not cause morphological changes in HEK293 cells (Human Embryonic Kidney 293 cells) (Figure 3B). Living cells were examined on a Leica TCS SP5 confocal microscope on a Leica DMI 6000 CS stand. We used a 63x oil immersion lens. Images were obtained with LAS AF 1.8.0. Leica Microsystems.
Primer 6: Produkcija fuzijskega proteina med elastinu podobnim segmentom in funkcionalno polipeptidno domeno, ki tvori srebrne nanodelce z protimikrobno aktivnostjo Konstrukt, ki je bil pripravljen z namenom prikaza produkcije proteina s funkcionalno polipeptidno domeno, ki povzroči nastanek srebrnih nanodelcev, je naveden v Tabeli 1 (No. 7). Produkcija in izolacija proteina je enaka kot v primeru 2. -30-Example 6: Production of a fusion protein between an elastin-like segment and a functional polypeptide domain forming silver nanoparticles with antimicrobial activity A construct prepared to show the production of a protein with a functional polypeptide domain causing silver nanoparticles is given in Table 1 ( No. 7). Protein production and isolation is the same as in case 2. -30-
Primer 7: Protimikrobno delovanje polipeptidnega materiala, ki vsebuje funkcionalno polipeptidno domeno za tvorbo srebrnih nanodelcev AEA je načrtovano trimerizacijsko zaporedje, za tvorbo ovite vijačnice (SEQ ID: 26). Sestavljeno je iz treh segmentov, ki se združijo v stopničasti heterotrimer in imajo na mestih b, c and/heptade ovite vijačnice aminokislinske ostanke Ala (b), Ala (c) and Glu (/). AEA katalizira nastanek srebrnih nanodelcev iz ionov srebra v raztopini. AEA združen z elastinu podobnim segmentom (priprava je opisana v primeru 6) je del polipeptidnega materiala. AEA segment materiala reducira Ag+ v Ag° v obliki nanodelcev. Po odstranitvi nereduciranih ionov srebra z dializo srebrni nanodelci ostanejo pritrjeni na matriks.Example 7: Antimicrobial activity of a polypeptide material containing a functional polypeptide domain for the formation of AEA silver nanoparticles is a planned trimerization sequence for the formation of a coiled helix (SEQ ID: 26). It consists of three segments that combine into a stepped heterotrimer and have the amino acid residues Ala (b), Ala (c) and Glu (/) at the b, c and / heptade helix sites. AEA catalyzes the formation of silver nanoparticles from silver ions in solution. The AEA associated with the elastin-like segment (preparation described in Example 6) is part of the polypeptide material. The AEA material segment reduces Ag + to Ag ° in the form of nanoparticles. After removal of unreduced silver ions by dialysis, the silver nanoparticles remain attached to the matrix.
Pripravili smo štiri različne mešanice (Tabela 3) z bakterijami (E. coli DC2). Po prekonočni inkubaciji (37°C) smo izmerili OD pri 600 nm (Nano Drop). Vrednosti OD600 mešanic 1 in 3 je prikazan na sliki 4.Four different mixtures (Table 3) with bacteria (E. coli DC2) were prepared. After overnight incubation (37 ° C), OD was measured at 600 nm (Nano Drop). The OD600 values of mixtures 1 and 3 are shown in Figure 4.
Tabela 3: Mešanice, ki so bile pripravljene za preverjanje protimikrobnega delovanja polipeptidnega materiala, ki vsebuje funkcionalno polipeptidno domeno za tvorbo srebrnih nanodelcev, in srebrovega acetata Št. mešanica 1 dializiran AEA-Ag + LB + £. coli DC2 2 dializiran AEA-Ag + LB (brez bakterij) 3 dializiran Ag-acetat + LB + E. coli DC2 4 dializiran Ag-acetat + LB (brez bakterij)Table 3: Mixtures prepared for testing the antimicrobial activity of a polypeptide material containing a functional polypeptide domain for the formation of silver nanoparticles and silver acetate mixture 1 dialyzed AEA-Ag + LB + £. coli DC2 2 dialyzed AEA-Ag + LB (no bacteria) 3 dialyzed Ag-acetate + LB + E. coli DC2 4 dialyzed Ag-acetate + LB (no bacteria)
Primer 8: Nagnjenost k tvorbi heterodimerov segmentov za tvorbo ovite vijačnice Pl in P2 Segmente za tvorbo ovitih vijačnic Pl do P8 so izoblikovali predlagatelji. Pari (P1-P2, P3-P4, P5-P6, P7-P8) tvorijo α-vijačnico le v primeru, da sta prisotni obe komponenti. Princip je prikazan na paru P1-P2. Peptida Pl in P2 sta bila bila sintetizirana s sintezo s trdno fazo v Keck centru, Yale University, New Heaven, ZDA. Sintetični segment za tvorbo ovite vijačnice Pl je bil raztopljen v 0.1% amonijevem bikarbonatu s končno koncentracijo 5 mg/ml. P2 je bil raztopljen v destilirani vodi s končno koncentracijo 5 mg/ml. CD spekter je bil posnet pod pretokom dušika z AppliedPhotophysics Chirascan spektropolarimetrom (Applied Photophysics, Surrey, UK). CD spektre v daljnjem UV območju posameznih segmentov za tvorbo ovitih vijačnic in njihovih mešanic smo merili v celici z optično potjo 0.1 cm vsakih 0.5 nm v območju valovnih dolžin 190nm- 260nm, pri čemer je bil -31- čas merjenja pri eni valovni dolžini 1 s. Koncentracija segmentov, za tvorbo ovitih vijačnic, je bila 0.1 mg/ml. CD spekter posameznih segmentov za tvorbo ovitih vijačnic (Pl in P2), kaže naključno strukturo proteina. V primeru, ko sta bila segmenta za tvorbo ovite vijačnice, zmešana, spekter nakazuje α-helikalno strukturo (Slika 5).Example 8: The tendency to form heterodimers of the segments for the formation of the wrapped helix Pl and P2 The segments for the formation of the wrapped helix Pl to P8 were designed by the proponents. Pairs (P1-P2, P3-P4, P5-P6, P7-P8) form an α-helix only if both components are present. The principle is shown on the pair P1-P2. Peptides Pl and P2 were synthesized by solid phase synthesis at the Keck Center, Yale University, New Heaven, USA. The synthetic segment for the formation of the wrapped helix Pl was dissolved in 0.1% ammonium bicarbonate with a final concentration of 5 mg / ml. P2 was dissolved in distilled water at a final concentration of 5 mg / ml. The CD spectrum was recorded under nitrogen flow with an AppliedPhotophysics Chirascan spectropolarimeter (Applied Photophysics, Surrey, UK). CD spectra in the further UV range of individual segments for the formation of wrapped helices and their mixtures were measured in a cell with an optical path of 0.1 cm every 0.5 nm in the wavelength range 190nm-260nm, where -31- was the measurement time at one wavelength 1 s . The concentration of segments, for the formation of wrapped helices, was 0.1 mg / ml. The CD spectrum of individual segments for the formation of coiled helices (Pl and P2) shows the random structure of the protein. In the case where the two segments for the formation of the coiled helix were mixed, the spectrum indicates an α-helical structure (Figure 5).
Primer 9: Pridobivanje celic iz polipeptidnega materiala Z dodatkom segmenta za tvorbo ovitih vijačnic in je zmožen oligomerizacije s tvorbo ovite vijačnice z enim od segmentov polipeptidnega materiala, se polipeptidni material enostavno razstavi. Dodani segment depolimerizira polipeptidni material tudi s tem, da se veže na segmente za tvorbo ovitih vijačnic in so spojeni s funkcionalnimi polipeptidnimi domenami. Celice se tako nežno sprostijo iz matriksa, brez uporabe ostrih fizikalnih pogojev kot so temperatura, ozmotski šok, ionska jakost ali encimska razgradnja, ki vplivajo na ali poškodujejo površinske receptorje in adhezijske molekule.Example 9: Obtaining Cells from Polypeptide Material By adding a wrapped helix segment and capable of oligomerization by forming a wrapped helix with one of the polypeptide material segments, the polypeptide material is easily disassembled. The added segment also depolymerizes the polypeptide material by binding to the segments to form coiled helices and are fused to functional polypeptide domains. The cells are thus gently released from the matrix, without the use of harsh physical conditions such as temperature, osmotic shock, ionic strength, or enzymatic degradation that affect or damage surface receptors and adhesion molecules.
Polipeptidni matriks je bil sestavljen iz fuzijskih proteinov naštetih v Tabeli 1 (SEQ ID NO: 6, 8). Po polimerizaciji je bil rezultat dodatka 50-kranega molamega presežka Pl ali P2 razgradnja polipeptidnega materiala. -32- ««·· • ·The polypeptide matrix was composed of fusion proteins listed in Table 1 (SEQ ID NO: 6, 8). After polymerization, the addition of a 50-fold molar excess of Pl or P2 resulted in degradation of the polypeptide material. -32- «« ·· • ·
ZAPOREDJA <210> 1 <211> 960 <212> DNK <213> Sintetični <220> <221> CDS <222> (1)..(960) <223> KSI-DP-Histag-LL37-ELST-GCN- ELST <400> 1 atg cat acc cca gaa cac ate acc gcc gtg gta cag cgc ttt gtg gct 48 Met His Thr Pro Glu His Ile Thr Ala Val Val Gin Arg Phe Val Ala 1 5 10 15 gcg ctc aat gcc gg° gat ctg gac ggc ate gtc gcg ctg ttt gcc gat 96 Al a Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp 20 25 30 gac gcc acg gtg gaa gac ccc gtg ggt tcc gag ccc agg tcc ggt acg 144 Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr 35 40 45 gct gcg att cgt gag ttt tac gcc aac teg ctc aaa ctg cct ttg gcg 192 Ala Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala 50 55 60 gtg gag ctg acg cag gag gta cgc gcg gtc gcc aac gaa gcg gcc ttc 240 Val Glu Leu Thr Gin Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe 65 70 75 80 gct ttc acc gtc agc ttc gag tat cag ggc cgc aag acc gta gtt gcg 288 Ala Phe Thr Val Ser Phe Glu Tyr Gin Gly Arg Lys Thr Val Val Ala 85 90 95 ccc ate gat cac ttt cgc ttc aat ggc gcc ggc aag gtg gtg agc ate 336 Pro 11 e Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile 100 105 110 cgc gcc ttg ttt ggc gag aag aat att cac gca tgc cag atg gat cct 384 Arg Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gin Met Asp Pro 115 120 125 atg tat cat cac cat cac cat cac tet aga gcc ggc ctg ctg ggt gat 432 Met Tyr His His His His His His Ser Arg Ala Gly Leu Leu Gly Asp 130 135 140 ttc ttc cgg aaa tet aaa gag aag att ggc aaa gag ttt aaa aga att 480 Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu Phe Lys Arg Ile 145 150 155 160 gtc cag aga ate aag gat ttt ttg cgg aat ctt gta ccc agg aca gag 528 Val Gin Arg Ile Lys Asp Phe Leu Arg Asn Leu Val Pro Arg Thr Glu 165 170 175 tcc tcc ggg gtt ccg ggt gtt ggt gtt cct ggc gtg ggt gtt cct ggt 576 Ser Ser Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 180 185 190 624 624 gtt ggc gtg cca ggt gtg gga gtg cca ggc gtt ggt gta ccg ggt gtg Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 195 200 205 ggc gtt ccg gga gtc gga gtt ccg gga gta ggc gtg cct ggt gtt ggg Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 210 215 220 gtt ccg ggt gta ggt tcc ggg cgt atg aaa cag ctg gaa gat aaa ate Val Pro Gly Val Gly Ser Gly Arg Met Lys Gin Leu Glu Asp Lys Ile 225 230 235 240 gag gag ctg ctg tet aag ate tac cac ctg gaa aac gaa att get ege Glu Glu Leu Leu Ser Lys Ile Tyr His Leu Glu Asn Glu Ile Ala Arg 245 250 255 ctg aaa aag ctg att ggt gaa ege tcc ggg gtt ccg ggt gtt ggt gtt Leu Lys Lys Leu Ile Gly Glu Arg Ser Gly Val Pro Gly Val Gly Val 260 265 270 cct ggc gtg ggt gtt cct ggt gtt ggc gtg cca ggt gtg gga gtg cca Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 275 280 285 ggc gtt ggt gta ccg ggt gtg ggc gtt ccg gga gtc gga gtt ccg gga Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 290 295 300 gta ggc gtg cct ggt gtt ggg gtt ccg ggt gta ggt tcc gga act agt Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Ser Gly Thr Ser 305 310 315 * 320 <210> 2 <211> 320 <212> PRT <213> Sintetični <400> 2 Met His Thr Pro Glu His Ile Thr Ala Val Val Gin Arg Phe Val Ala 1 5 10 15 Al a Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp 20 25 30 Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr 35 40 45 Al a Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala 50 55 60 Val Glu Leu Thr Gin Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe 65 70 75 80 Ala Phe Thr Val Ser Phe Glu Tyr Gin Gly Arg Lys Thr Val Val Ala 85 90 95 Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile 100 105 110 672 720 768 816 864 912 960SEQUENCE < 210 > 1 < 211 > 960 < 212 > DNA < 213 > Synthetic < 220 > < 221 > CDS < 222 > (1) .. (960) < 223 > KSI-DP-Histag-LL37-ELST-GCN-ELST < 400 > 1 atg cat acc cca gaa cac ate acc gcc gtg gta cag cgc ttt gtg gct 48 Met His Thr Pro Glu His Ile Thr Ala Val Val Gin Arg Phe Val Ala 1 5 10 15 gcg ctc aat gcc gg ° gat ctg gac ggc ate gtc gcg ctg ttt gcc gat 96 Al a Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp 20 25 30 gac gcc acg gtg gaa gac ccc gtg ggt tcc gag ccc agg tcc ggt acg 144 Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr 35 40 45 gct gcg att cgt gag ttt tac gcc aac teg ctc aaa ctg cct ttg gcg 192 Ala Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala 50 55 60 gtg gag ctg acg cag gag gta cgc gcg gtc gcc aac gaa gcg gcc ttc 240 Val Glu Leu Thr Gin Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe 65 70 75 80 gct ttc acc gtc agc ttc gag tat cag ggc cgc aag acc gta gtt gcg 288 Ala Phe Thr Val Ser Phe Glu Tyr Gin Gly Arg Lys Thr Val Val Ala 85 90 95 ccc ate gat cac ttt cgc ttc aat ggc gcc ggc aag gtg gtg agc ate 336 Dec 11 e Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile 100 105 110 cgc gcc ttg ttt ggc gag aag aat att cac gca tgc cag atg gat cct 384 Arg Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gin Met Asp Pro 115 120 125 atg tat cat cac cat cac cat cac tet aga gcc ggc ctg ctg ggt gat 432 Met Tyr His His His His His Ser Arg Ala Gly Leu Leu Gly Asp 130 135 140 ttc ttc cgg aaa tet aaa gag aag att ggc aaa gag ttt aaa aga att 480 Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu Phe Lys Arg Ile 145 150 155 160 gtc cag aga ate aag gat ttt ttg cgg aat ctt gta ccc agg gag 528 Val Gin Arg Ile Lys Asp Phe Leu Arg Asn Leu Val Pro Arg Thr Glu 165 170 175 tcc tcc ggg gtt ccg ggt gtt ggt gtt cct ggc gtg ggt gtt cct ggt 576 Ser Ser Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 180 185 190 624 624 gtt ggc gtg cca ggt gtg gga gtg cca ggc gtt ggt gta ccg ggt g tg Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 195 200 205 ggc gtt ccg gga gtc gga gtt ccg gga gta ggc gtg cct ggt gtt ggg Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 210 215 220 gtt ccg ggt gta ggt tcc ggg cgt atg aaa cag ctg gaa gat aaa ate Val Pro Gly Val Gly Ser Gly Arg Met Lys Gin Leu Glu Asp Lys Ile 225 230 235 240 gag gag ctg ctg tet aag ate tac cac ctg gaa aac gaa att get ege Glu Glu Leu Leu Ser Lys Ile Tyr His Leu Glu Asn Glu Ile Ala Arg 245 250 255 ctg aaa aag ctg att ggt gaa ege tcc ggg gtt ccg ggt gtt ggt gtt Leu Lys Lys Leu Arg Ile Gly Ser Gly Val Pro Gly Val Gly Val 260 265 270 cct ggc gtg ggt gtt cct ggt gtt ggc gtg cca ggt gtg gga gtg cca Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 275 280 285 ggc gtt ggt gta ccg ggt gtg ggc gtt ccg gga gtc gga gtt ccg gga Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val P ro Gly 290 295 300 gta ggc gtg cct ggt gtt ggg gtt ccg ggt gta ggt tcc gga act agt Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Ser Gly Thr Ser 305 310 315 * 320 < 210 > 2 < 211 > 320 < 212 > PRT < 213 > Synthetic < 400 > 2 Met His Thr Pro Glu His Ile Thr Ala Val Val Gin Arg Phe Val Ala 1 5 10 15 Al a Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp 20 25 30 Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr 35 40 45 Al a Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala 50 55 60 Val Glu Leu Thr Gin Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe 65 70 75 80 Ala Phe Thr Val Ser Phe Glu Tyr Gin Gly Arg Lys Thr Val Val Ala 85 90 95 Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile 100 105 110 672 720 768 816 864 912 960
Arg Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gin Met Asp Pro 115 120 125 Met Tyr His His His His His His Ser Arg Ala Gly Leu Leu Gly Asp 130 135 140 Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu Phe Lys Arg Ile 145 150 155 160 Val Gin Arg Ile Lys Asp Phe Leu Arg Asn Leu Val Pro Arg Thr Glu 165 170 175 Ser Ser Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 180 185 190 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 195 200 205 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 210 215 220 Val Pro Gly Val Gly Ser Gly Arg Met Lys Gin Leu Glu Asp Lys Ile 225 230 235 240 Glu Glu Leu Leu Ser Lys Ile Tyr His Leu Glu Asn Glu Ile Ala Arg 245 250 255 Leu Lys Lys Leu Ile Gly Glu Arg Ser Gly Val Pro Gly Val Gly Val 260 265 270 Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 275 280 285 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 290 295 300 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Ser Gly Thr Ser 305 310 315 320 <210> 3 <211> 1740 <212> DNK <213> Sintetični <220> <221> CDS <222> (D · .(1740) <223> KSI-1 DP-Histag-LL37-ELST-GCN-l P2-ELST-mNGF <400> 3 atg cat acc cca gaa cac ate acc gcc gtg gta cag ege ttt gtg get Met His Thr Pro Glu His Ile Thr Ala Val Val Gin Arg Phe Val Ala 1 5 10 15 gcg ctc aat gcc ggc gat ctg gac ggc ate gtc gcg ctg ttt gcc gat Ala Leu As n Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp 20 25 30 gac gcc acg gtg gaa gac ccc gtg ggt tcc gag ccc agg tcc ggt acg Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr 48 96 144 -35- 35 40 45 gct gcg att cgt gag ttt tac gcc aac teg ete aaa ctg cct ttg gcg 192 Al a Al a Ile Arg Glu Phe Tyr Al a Asn Ser Leu Lys Leu Pro Leu Ala 50 55 60 gtg gag ctg acg cag gag gta cgc gcg gtc gcc aac gaa gcg gcc ttc 240 Val Glu Leu Thr Gin Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe 65 70 75 80 gct ttc acc gtc agc ttc gag tat cag ggc cgc aag acc gta gtt gcg 288 Al a Phe Thr Val Ser Phe Glu Tyr Gin Gly Arg Lys Thr Val Val Ala 85 90 95 ccc ate gat cac ttt cgc ttc aat ggc gcc ggc aag gtg gtg agc ate 336 Pro Ile Asp His Phe Arg Phe As n Gly Ala Gly Lys Val Val Ser Ile 100 105 110 cgc gcc ttg ttt ggc gag aag aat att cac gca tgc cag atg gat cct 384 Arg Al a Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gin Met Asp Pro 115 120 125 atg tat cat cac cat cac cat cac tet aga gcc ggc ctg ctg ggt gat 432 Met Tyr His His His His His His Ser Arg Ala Gly Leu Leu Gly Asp 130 135 140 ttc ttc cgg aaa tet aaa gag aag att ggc aaa gag ttt aaa aga att 480 Phe Phe Arg Lys Ser Lys Glu Ly s Ile Gly Lys Glu Phe Lys Arg Ile 145 150 155 160 gtc cag aga ate aag gat ttt ttg cgg aat ctt gta ccc agg aca gag 528 Val Gin Arg Ile Lys Asp Phe Leu Arg Asn Leu Val Pro Arg Thr Glu 165 170 175 tcc tcc ggg gtt ccg ggt gtt ggt gtt cct ggc gtg ggt gtt cct ggt 576 Ser Ser Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 180 185 190 gtt ggc gtg cca ggt gtg gga gtg cca ggc gtt ggt gta ccg ggt gtg 624 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 195 200 205 ggc gtt ccg gga gtc gga gtt ccg gga gta ggc gtg cct ggt gtt ggg 672 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 210 215 220 gtt ccg ggt gta ggt tcc ggg cgt atg aaa cag ctg gaa gat aaa ate 720 Val Pro Gly Val Gly Ser Gly Arg Met Lys Gin Leu Glu Asp Lys Ile 225 230 235 240 gag gag ctg ctg tet aag ate tac cac ctg gaa aac gaa att gct cgc 768 Glu Glu Leu Leu Ser Lys Ile Tyr His Leu Glu Asn Glu Ile Ala Arg 245 250 255 ctg aaa aag ctg att ggt gaa cgc tcc ggg tet cca gaa gac aaa ate 816 Leu Lys Lys Leu Ile Gly Glu Arg Ser Gly Ser Pro Glu Asp Lys Ile 260 265 270 gca cag ctg aaa gaa aag aac gcg gcc ctg aaa gaa aag aat caa cag 864 Al a Gin Leu Lys Glu Lys As n Al a Ala Leu Lys Glu Lys Asn Gin Gin 275 280 285 -36- ctg aag gag aaa ate caa gca ctg aaa tat ggc tcc ggg gtt ccg ggt 912 Leu Lys Glu Lys Ile Gin Ala Leu Lys Tyr Gly Ser Gly Val Pro Gly 290 295 300 gtt ggt gtt cct ggc gtg ggt gtt cct ggt gtt ggc gtg cca ggt gtg 960 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 305 310 315 320 gga gtg cca ggc gtt ggt gta ccg ggt gtg ggc gtt ccg gga gtc gga 1008 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 325 330 335 gtt ccg gga gta ggc gtg cct ggt gtt ggg gtt ccg ggt gta ggt tcc 1056 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Ser 340 345 350 ggg gaa ccg tat act gat agc aac gtg cca gaa ggc gat agc gta cca 1104 Gly Glu Pro Tyr Thr Asp Ser Asn Val Pro Glu Gly Asp Ser Val Pro 355 360 365 gaa gcc cat tgg aca aaa ctg caa cac agc ctg gat aca gca ctg cgt 1152 Glu Al a His Trp Thr Lys Leu Gin His Ser Leu Asp Thr Ala Leu Arg 370 375 380 cgt gcc cgt tca gca cca aca get cct att get gcc cgt gtt act ggt 1200 Arg Al a Arg Ser Ala Pro Thr Ala Pro Ile Ala Ala Arg Val Thr Gly 385 390 395 400 cag act cgt aat ate acg gtg gat cct cgt ctg ttc aaa aaa ege ege 1248 Gin Thr Arg Asn Ile Thr Val Asp Pro Arg Leu Phe Lys Lys Arg Arg 405 410 415 ctg cat tet cct cgt gtt ctg ttt agc aca caa cct ccg cca aca tca 1296 Leu His Ser Pro Arg Val Leu Phe Ser Thr Gin Pro Pro Pro Thr Ser 420 425 430 agc gat aca ctg gac ctg gac ttt caa gca cat gga acc att ccg ttc 1344 Ser As p Thr Leu Asp Leu Asp Phe Gin Ala His Gly Thr Ile Pro Phe 435 440 445 aat cgt acc cat cgt tca aaa ege tca agc acc cat ccg gtg ttt cat 1392 Asn Arg Thr His Arg Ser Lys Arg Ser Ser Thr His Pro Val Phe His 450 455 460 atg ggg gaa ttt teg gtt tgt gac agc gtg tet gtc tgg gtt ggg gat 1440 Met Gly Glu Phe Ser Val Cys Asp Ser Val Ser Val Trp Val Gly Asp 465 470 475 480 aaa acc aca gcg acc gat ate aaa ggc aaa gaa gtg acc gtc ctg gcc 1488 Lys Thr Thr Al a Thr Asp Ile Lys Gly Lys Glu Val Thr Val Leu Ala 485 490 495 gaa gtc aat ate aac aac agc gtc ttt cgt caa tat ttc ttc gaa acc 1536 Glu Val Asn Ile Asn Asn Ser Val Phe Arg Gin Tyr Phe Phe Glu Thr 500 505 510 aaa tgc cgt gcc tca aat cct gta gaa agc ggg tgt cgt gga att gat 1584 Lys Cys Arg Ala Ser Asn Pro Val Glu Ser Gly Cys Arg Gly Ile Asp 515 520 525 -37- • · · · tca aaa cat tgg aac tcg tat tgt acc acc act cac acc ttc gtt aaa 1632 Ser Lys His Trp Asn Ser Tyr Cys Thr Thr Thr His Thr Phe Val Lys 530 535 540 gcc ctg act acc gac gag aaa caa gct gct tgg cgc ttt ate cgt ate 1680 Ala Leu Thr Thr Asp Glu Lys Gin Ala Ala Trp Arg Phe Ile Arg Ile 545 550 555 560 gat acc gct tgt gtg tgt gtc ctg tcc cgt aaa gca aca cgt cgt ggt 1728 Asp Thr Ala Cys Val Cys Val Leu Ser Arg Lys Ala Thr Arg Arg Gly 565 570 575 tcc gga act agt 1740 Ser Gly Thr Ser 580 <210> 4 <211> 580 <212> PRT <213> Sintetični <4 00> 4Arg Ala Leu Phe Gly Glu Llu Asn Ile His Ala Cys Gin Met Asp Pro 115 120 125 Met Tyr His His His His His His Ser Arg Ala Gly Leu Leu Gly Asp 130 135 140 Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu Phe Lys Arg Ile 145 150 155 160 Val Gin Arg Ile Lys Asp Phe Leu Arg Asn Leu Val Pro Arg Thr Glu 165 170 175 Ser Ser Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 180 185 190 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 195 200 205 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 210 215 220 Val Pro Gly Val Gly Ser Gly Arg Met Lys Gin Leu Glu Asp Lys Ile 225 230 235 240 Glu Glu Leu Leu Ser Lys Ile Tyr His Leu Glu Asn Glu Ile Ala Arg 245 250 255 Leu Lys Lys Leu Ile Gly Glu Arg Ser Gly Val Pro Gly Val Gly Val 260 265 270 Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 275 280 285 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 290 295 300 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Ser Gly Thr Ser 305 310 315 320 < 210 > 3 < 211 > 1740 < 212 > DNA < 213 > Synthetic < 220 > < 221 > CDS < 222 > (D ·. (1740) < 223 > KSI-1 DP-Histag-LL37-ELST-GCN-l P2-ELST-mNGF < 400 > 3 atg cat acc cca gaa cac ate acc gcc gtg gta cag ege ttt gtg get Met His Thr Pro Glu His Ile Thr Ala Val Val Gin Arg Phe Val Ala 1 5 10 15 gcg ctc aat gcc ggc gat ctg gac ggc ate gtc gcg ctg ttt gcc gat Ala Leu As n Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp 20 25 30 gac gcc acg gtg gaa gac ccc gtg ggt tcc gag ccc agg tcc ggt acg Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr 48 96 144 -35- 35 40 45 gct gcg att cgt gag ttt tac gcc aac teg ete aaa ctg cct ttg gcg 192 Al a Al a Ile Arg Glu Phe Tyr Al a Asn Ser Leu Lys Leu Pro Leu Ala 50 55 60 gtg gag ctg acg cag gag gta cgc gcg gtc gcc aac gaa gcg gcc ttc 240 Val Glu Leu Thr Gin Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe 65 70 75 80 gct ttc acc gtc agc ttc gag tat cag ggc cgc aag acc gta gtt gcg 288 Al a Phe Thr Val Ser P he Glu Tyr Gin Gly Arg Lys Thr Val Val Ala 85 90 95 ccc ate gat cac ttt cgc ttc aat ggc gcc ggc aag gtg gtg agc ate 336 Pro Ile Asp His Phe Arg Phe As n Gly Ala Gly Lys Val Val Ser Ile 100 105 110 cgc gcc ttg ttt ggc gag aag aat att cac gca tgc cag atg gat cct 384 Arg Al a Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gin Met Asp Pro 115 120 125 atg tat cat cac cat cac cat cac tet aga gcc ggc ctg ctg ggt gat 432 Met Tyr His His His His His His Ser Arg Ala Gly Leu Leu Gly Asp 130 135 140 ttc ttc cgg aaa tet aaa gag aag att ggc aaa gag ttt aaa aga att 480 Phe Phe Arg Lys Ser Lys Glu Ly s Ile Gly Lys Glu Phe Lys Arg Ile 145 150 155 160 gtc cag aga ate aag gat ttt ttg cgg aat ctt gta ccc agg aca gag 528 Val Gin Arg Ile Lys Asp Phe Leu Arg Asn Leu Val Pro Arg Thr Glu 165 170 175 tcc tcc ggg gtt ccg ggt gtt ggt gtt cct ggc gtg ggt gtt cct ggt 576 Ser Ser Gly Val Pro Gly Val Gly Val Pro Gly Va l Gly Val Pro Gly 180 185 190 gtt ggc gtg cca ggt gtg gga gtg cca ggc gtt ggt gta ccg ggt gtg 624 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 195 200 205 ggc gtt ccg gga gtc gga gtt ccg gga gta ggc gtg cct ggt gtt ggg 672 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 210 215 220 gtt ccg ggt gta ggt tcc ggg cgt atg aaa cag ctg gaa gat aaa ate 720 Val Pro Gly Val Gly Ser Gly Arg Met Lys Gin Leu Glu Asp Lys Ile 225 230 235 240 gag gag ctg ctg tet aag ate tac cac ctg gaa aac gaa att gct cgc 768 Glu Glu Leu Leu Ser Lys Ile Tyr His Leu Glu Asn Glu Ile Ala Arg 245 250 255 ctg aaa aag ctg att ggt gaa cgc tcc ggg tet cca gaa gac aaa ate 816 Leu Lys Lys Leu Ile Gly Glu Arg Ser Gly Ser Pro Glu Asp Lys Ile 260 265 270 gca cag ctg aaa gaa aag aac gcg gcc ccc gaa aag aat caa cag 864 Al a Gin Leu Lys Glu Lys As n Al a Ala Leu Lys Glu Lys Asn Gin Gin 275 280 285 -36- ctg aag gag aaa ate caa gca ctg aaa tat ggc tcc ggg gtt ccg ggt 912 Leu Lys Glu Lys Ile Gin Ala Leu Lys Tyr Gly Ser Gly Val Pro Gly 290 295 300 gtt ggt gtt cct ggc gtg gtg cct ggt gtt ggc gtg cca ggt gtg 960 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 305 310 315 320 gga gtg cca ggc gtt ggt gta ccg ggt gtg ggc gtt ccg gga gtc gga 1008 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 325 330 335 gtt ccg gga gta ggc gtg cct ggt gtt ggg gtt ccg ggt gta ggt tcc 1056 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Ser 340 345 350 ggg gaa ccg tat act gat agc aac gtg cca gaa ggc gat agc gta cca 1104 Gly Glu Pro Tyr Thr Asp Ser Asn Val Pro Glu Gly Asp Ser Val Pro 355 360 365 gaa gcc cat tgg aca aaa ctg caa cac agc ctg gat aca gca ctg cgt 1152 Glu Al a His Trp Thr Lys Leu Gin His Ser Leu Asp Thr Ala Leu Arg 370 375 380 cgt gcc cgt tca gca cca aca get cct att get gcc cgt gtt act ggt 1200 Arg Al a Arg Ser Ala Pro Thr Ala Pro Ile Ala Ala Arg Val Thr Gly 385 390 395 400 cag act cgt aat ate acg gtg gat cct cgt ctg ttc aaa aaa ege ege 1248 Gin Thr Arg Asn Ile Thr Val Asp Pro Arg Leu Phe Lys Lys Lys Arg Arg 405 410 415 ctg cat tet cct cgt gtt ctg ttt agc aca caa cct ccg cca aca tca 1296 Leu His Ser Pro Arg Val Leu Phe Ser Thr Gin Pro Pro Pro Thr Ser 420 425 430 agc gat aca ctg gac ctg gac ttt caa gca cat gga acc att ccg ttc 1344 Ser As p Thr Leu Asp Leu Asp Phe Gin Ala His Gly Thr Ile Pro Phe 435 440 445 aat cgt acc cat cgt tca aaa ege tca agc acc cat ccg gtg ttt cat 1392 Asn Arg Thr His Arg Ser Lys Arg Ser Ser Thr His Pro Val Phe His 450 455 460 atg ggg gaa ttt teg gtt tgt gac agc gtg tet gtc tgg gtt ggg gat 1440 Met Gly Glu Phe Ser Val Cys Asp Ser Val Ser Val Trp Val Gly Asp 465 470 475 480 aaa acc ac a gcg acc gat ate aaa ggc aaa gaa gtg acc gtc ctg gcc 1488 Lys Thr Thr Al a Thr Asp Ile Lys Gly Lys Glu Val Thr Val Leu Ala 485 490 495 gaa gtc aat ate aac aac agc gtc ttt cgt caa tat ga ttc acc 1536 Glu Val Asn Ile Asn Asn Ser Val Phe Arg Gin Tyr Phe Phe Glu Thr 500 505 510 aaa tgc cgt gcc tca aat cct gta gaa agc ggg tgt cgt gga att gat 1584 Lys Cys Arg Ala Ser Asn Pro Val Glu Ser Gly Cys Arg Gly Ile Asp 515 520 525 -37- • · · · tca aaa cat tgg aac tcg tat tgt acc acc act act cac acc ttc gtt aaa 1632 Ser Lys His Trp Asn Ser Tyr Cys Thr Thr Thr His Thr Phe Val Lys 530 535 540 gcc ctg act acc gac gag aaa caa gct gct tgg cgc ttt ate cgt ate 1680 Ala Leu Thr Thr Asp Glu Lys Gin Ala Ala Trp Arg Phe Ile Arg Ile 545 550 555 560 gat acc gct tgt gtg tgt gtc ctg tcc cgca a cgt cgt ggt 1728 Asp Thr Ala Cys Val Cys Val Leu Ser Arg Lys Ala Thr Arg Arg Gly 565 570 575 tcc gga act agt 1740 Ser Gly Thr Ser 580 < 210 > 4 < 211 > 580 < 212 > PRT < 213 > Synthetic < 4 00 > 4
Met His Thr Pro Glu His Ile Thr Ala Val Val Gin Arg Phe Val Ala 1 5 10 15 Ala Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp 20 25 30 Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr 35 40 45 Ala Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala 50 55 60 Val Glu Leu Thr Gin Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe 65 70 75 80 Ala Phe Thr Val Ser Phe Glu Tyr Gin Gly Arg Lys Thr Val Val Ala 85 90 95 Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile 100 105 110 Arg Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gin Met Asp Pro 115 120 125 Met Tyr His His His His His His Ser Arg Ala Gly Leu Leu Gly Asp 130 135 140 Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu Phe Lys Arg Ile 145 150 155 160 Val Gin Arg Ile Lys Asp Phe Leu Arg Asn Leu Val Pro Arg Thr Glu 165 170 175 Ser Ser Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 180 185 190Met His Thr Pro Glu His Ile Thr Ala Val Val Gin Arg Phe Val Ala 1 5 10 15 Ala Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp 20 25 30 Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr 35 40 45 Ala Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala 50 55 60 Val Glu Leu Thr Gin Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe 65 70 75 80 Ala Phe Thr Val Ser Phe Glu Tyr Gin Gly Arg Lys Thr Val Val Ala 85 90 95 Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile 100 105 110 Arg Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gin Met Asp Pro 115 120 125 Met Tyr His His His His His Ser Arg Ala Gly Leu Leu Gly Asp 130 135 140 Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu Phe Lys Arg Ile 145 150 155 160 Val Gin Arg Ile Lys Asp Phe Leu Arg Asn Leu Val Pro Arg Thr Glu 165 170 175 Ser Ser Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 180 185 190
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val -38- ··*· 195 200 205 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 210 215 220 Val Pro Gly Val Gly Ser Gly Arg Met Lys Gin Leu Glu Asp Lys Ile 225 230 235 240 Glu Glu Leu Leu Ser Lys Ile Tyr His Leu Glu Asn Glu Ile Ala Arg 245 250 255 Leu Lys Lys Leu Ile Gly Glu Arg Ser Gly Ser Pro Glu Asp Lys Ile 260 265 270 Ala Gin Leu Lys Glu Lys Asn Ala Ala Leu Lys Glu Lys Asn Gin Gin 275 280 285 Leu Lys Glu Lys Ile Gin Ala Leu Lys Tyr Gly Ser Gly Val Pro Gly 290 295 300 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 305 310 315 320 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 325 330 335 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Ser 340 345 350 Gly Glu Pro Tyr Thr Asp Ser Asn Val Pro Glu Gly Asp Ser Val Pro 355 360 365 Glu Ala His Trp Thr Lys Leu Gin His Ser Leu Asp Thr Ala Leu Arg 370 375 380 Ar g Ala Arg Ser Ala Pro Thr Ala Pro Ile Ala Ala Arg Val Thr Gly 385 390 395 400 Gin Thr Arg Asn Ile Thr Val Asp Pro Arg Leu Phe Lys Lys Arg Arg 405 410 415 Leu His Ser Pro Arg Val Leu Phe Ser Thr Gin Pro Pro Pro Thr Ser 420 425 430 Ser As p Thr Leu Asp Leu Asp Phe Gin Ala His Gly Thr Ile Pro Phe 435 440 445 Asn Arg Thr His Arg Ser Lys Arg Ser Ser Thr His Pro Val Phe His 450 455 460 Met Gly Glu Phe Ser Val Cys Asp Ser Val Ser Val Trp Val Gly Asp 465 470 475 480 Lys Thr Thr Ala Thr Asp Ile Lys Gly Lys Glu Val Thr Val Leu Ala 485 490 495 Glu Val Asn Ile Asn Asn Ser Val Phe Arg Gin Tyr Phe Phe Glu Thr 500 505 510 Lys Cys Arg Ala Ser Asn Pro Val Glu Ser Gly Cys Arg Gly Ile Asp 515 520 525Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val -38- ·· * · 195 200 205 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 210 215 220 Val Pro Gly Val Gly Ser Gly Arg Met Lys Gin Leu Glu Asp Lys Ile 225 230 235 240 Glu Glu Leu Leu Ser Lys Ile Tyr His Leu Glu Asn Glu Ile Ala Arg 245 250 255 Leu Lys Lys Leu Ile Gly Glu Arg Ser Gly Ser Pro Glu Asp Lys Ile 260 265 270 Ala Gin Leu Lys Glu Lys Asn Ala Ala Leu Lys Glu Lys Asn Gin Gin 275 280 285 Leu Lys Glu Lys Ile Gin Ala Leu Lys Tyr Gly Ser Gly Val Pro Gly 290 295 300 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 305 310 315 320 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 325 330 335 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Ser 340 345 350 Gly Glu Pro Tyr Thr Asp Ser Asn Val Pro Glu Gly Asp Ser Val Pro 355 36 0 365 Glu Ala His Trp Thr Lys Leu Gin His Ser Leu Asp Thr Ala Leu Arg 370 375 380 Ar g Ala Arg Ser Ala Pro Thr Ala Pro Ile Ala Ala Arg Val Thr Gly 385 390 395 400 Gin Thr Arg Asn Ile Thr Val Asp Pro Arg Leu Phe Lys Lys Arg Arg 405 410 415 Leu His Ser Pro Arg Val Leu Phe Ser Thr Gin Pro Pro Pro Thr Ser 420 425 430 Ser As p Thr Leu Asp Leu Asp Phe Gin Ala His Gly Thr Ile Pro Phe 435 440 445 Asn Arg Thr His Arg Ser Lys Arg Ser Ser Thr His Pro Val Phe His 450 455 460 Met Gly Glu Phe Ser Val Cys Asp Ser Val Ser Val Trp Val Gly Asp 465 470 475 480 Lys Thr Thr Ala Thr Asp Ile Lys Gly Lys Glu Val Thr Val Leu Ala 485 490 495 Glu Val Asn Ile Asn Asn Ser Val Phe Arg Gin Tyr Phe Phe Glu Thr 500 505 510 Lys Cys Arg Ala Ser Asn Pro Val Glu Ser Gly Cys Arg Gly Ile Asp 515 520 525
Ser Lys His Trp As n Ser Tyr Cys Thr Thr Thr His Thr Phe Val Lys 530 535 540 Ala Leu Thr Thr Asp Glu Lys Gin Ala Ala Trp Arg Phe Ile Arg Ile 545 550 555 560 Asp Thr Ala Cys Val Cys Val Leu Ser Arg Lys Ala Thr Arg Arg Gly 565 570 575Ser Lys His Trp As n Ser Tyr Cys Thr Thr Thr His Thr Phe Val Lys 530 535 540 Ala Leu Thr Thr Asp Glu Lys Gin Ala Ala Trp Arg Phe Ile Arg Ile 545 550 555 560 Asp Thr Ala Cys Val Cys Val Leu Ser Arg Lys Ala Thr Arg Arg Gly 565 570 575
Ser Gly Thr Ser 580 <210> 5 <211> 1065 <212> DNK <213> Sintetični <220> <221> CDS <222> (1)..(1065) <223> KSI-DP-Histag-LL37-ELST-GCN- Pl-ELST <400> 5 atg cat acc cca gaa cac ate acc gcc gtg gta cag cgc ttt gtg gct 48 Met His Thr Pro Glu His Ile Thr Ala Val Val Gin Arg Phe Val Ala 1 5 10 15 gcg ctc aat gcc ggc gat ctg gac ggc ate gtc gcg ctg ttt gcc gat 96 Ala Leu As n Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp 20 25 30 gac gcc acg gtg gaa gac ccc gtg ggt tcc gag ccc agg tcc ggt acg 144 Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr 35 40 45 gct gcg att cgt gag ttt tac gcc aac teg ctc aaa ctg cct ttg gcg 192 Ala Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala 50 55 60 gtg gag ctg acg cag gag gta cgc gcg gtc gcc aac gaa gcg gcc ttc 240 Val Glu Leu Thr Gin Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe 65 70 75 80 gct ttc acc gtc agc ttc gag tat cag ggc cgc aag acc gta gtt gcg 288 Ala Phe Thr Val Ser Phe Glu Tyr Gin Gly Arg Lys Thr Val Val Ala 85 90 95 ccc ate gat cac ttt cgc ttc aat ggc gcc ggc aag gtg gtg agc ate 336 Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile 100 105 110 cgc gcc ttg ttt ggc gag aag aat att cac gca tgc cag atg gat cct 384 Arg Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gin Met Asp Pro 115 120 125 atg tat cat cac cat cac cat cac tet aga gcc ggc ctg ctg ggt gat 432 Met Tyr His His His His His His Ser Arg Ala Gly Leu Leu Gly Asp 130 135 140 ttc ttc cgg aaa tet aaa gag aag att ggc aaa gag ttt aaa aga att 480 -40- -40- Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu Phe Lys Arg Ile 145 150 155 160 gtc cag aga ate aag gat ttt ttg cgg aat ctt gta ccc agg aca gag 528 Val Gin Arg Ile Lys Asp Phe Leu Arg Asn Leu Val Pro Arg Thr Glu 165 170 175 tcc tcc ggg gtt ccg ggt gtt ggt gtt cct ggc gtg ggt gtt cct ggt 576 Ser Ser Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 180 185 190 gtt ggc gtg cca ggt gtg gga gtg cca ggc gtt ggt gta ccg ggt gtg 624 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 195 200 205 ggc gtt ccg gga gtc gga gtt ccg gga gta ggc gtg cct ggt gtt ggg 672 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 210 215 220 gtt ccg ggt gta ggt tcc ggg cgt atg aaa cag ctg gaa gat aaa ate 720 Val Pro Gly Val Gly Ser Gly Arg Met Lys Gin Leu Glu Asp Lys Ile 225 230 235 240 gag gag ctg ctg tet aag ate tac cac ctg gaa aac gaa att get ege 768 Glu Glu Leu Leu Ser Lys Ile Tyr His Leu Glu Asn Glu Ile Ala Arg 245 250 255 ctg aaa aag ctg att ggt gaa ege tcc ggg agc cca gaa gac gaa att 816 Leu Lys Lys Leu Ile Gly Glu Arg Ser Gly Ser Pro Glu Asp Glu Ile 260 265 270 cag gca ctg gaa gaa gaa aat get caa ctg gaa cag gaa aac gcg gcg 864 Gin Al a Leu Glu Glu Glu Asn Ala Gin Leu Glu Gin Glu Asn Ala Ala 275 280 285 ctg gaa gaa gaa ate gca cag ctg gaa tac ggc tcc ggg gtt ccg ggt 912 Leu Glu Glu Glu Ile Ala Gin Leu Glu Tyr Gly Ser Gly Val Pro Gly 290 295 300 gtt ggt gtt cct ggc gtg ggt gtt cct ggt gtt ggc gtg cca ggt gtg 960 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 305 310 315 320 gga gtg cca ggc gtt ggt gta ccg ggt gtg ggc gtt ccg gga gtc gga 1008 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 325 330 335 gtt ccg gga gta ggc gtg cct ggt gtt ggg gtt ccg ggt gta ggt tcc 1056 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Ser 340 345 350 gga act agt 1065Ser Gly Thr Ser 580 < 210 > 5 < 211 > 1065 < 212 > DNA < 213 > Synthetic < 220 > < 221 > CDS < 222 > (1) .. (1065) < 223 > KSI-DP-Histag-LL37-ELST-GCN-Pl-ELST < 400 > 5 atg cat acc cca gaa cac ate acc gcc gtg gta cag cgc ttt gtg gct 48 Met His Thr Pro Glu His Ile Thr Ala Val Val Gin Arg Phe Val Ala 1 5 10 15 gcg ctc aat gcc ggc gat ctg gac ggc ate gtc gcg ctg ttt gcc gat 96 Ala Leu As n Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp 20 25 30 gac gcc acg gtg gaa gac ccc gtg ggt tcc gag ccc agg tcc ggt acg 144 Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr 35 40 45 gct gcg att cgt gag ttt tac gcc aac teg ctc aaa ctg cct ttg gcg 192 Ala Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala 50 55 60 gtg gag ctg acg cag gag gta cgc gcg gtc gcc aac gaa gcg gcc ttc 240 Val Glu Leu Thr Gin Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe 65 70 75 80 gct ttc acc gtc agc ttc gag tat cag ggc cgc aag acc gta gtt gcg 288 Ala Phe Thr Val Ser Phe Glu Tyr Gin Gly Arg Lys Thr Val Val Ala 85 90 95 ccc ate gat cac ttt cgc ttc aat ggc gcc ggc aag gtg gtg agc ate 336 Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile 100 105 110 cgc gcc ttg ttt ggc gag aag aat att cac gca tgc cag atg gat cct 384 Arg Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gin Met Asp Pro 115 120 125 atg tat cat cac cat cac cat cac tet aga gcc ggc ctg ctg ggt gat 432 Met Tyr His His His His His Ser Arg Ala Gly Leu Leu Gly Asp 130 135 140 ttc ttc cgg aaa tet aaa gag aag att ggc aaa gag ttt aaa aga att 480 -40- -40- Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu Phe Lys Arg Ile 145 150 155 160 gtc cag aga ate aag gat ttt ttg cgg aat ctt gta ccc agg aca gag 528 Val Gin Arg Ile Lys Asp Phe Leu Arg Asn Leu Val Pro Arg Thr Glu 165 170 175 tcc tcc ggg gtt ccg ggt gtt ggt gtt cct ggc gtg ggt gtt cct ggt 576 Ser Ser Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 180 185 190 gtt ggc gtg cca ggt gtg gga gtg cca ggc gtt ggt gta ccg g gt gtg 624 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 195 200 205 ggc gtt ccg gga gtc gga gtt ccg gga gta ggc gtg cct ggt gtt ggg 672 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 210 215 220 gtt ccg ggt gta ggt tcc ggg cgt atg aaa cag ctg gaa gat aaa ate 720 Val Pro Gly Val Gly Ser Gly Arg Met Lys Gin Leu Glu Asp Lys Ile 225 230 235 240 gag gag ctg ctg tet aag ate tac cac ctg gaa aac gaa att get ege 768 Glu Glu Leu Leu Ser Lys Ile Tyr His Leu Glu Asn Glu Ile Ala Arg 245 250 255 ctg aaa aag ctg att ggt gaa ege tcc ggg agc cca gaa gac gaa att 816 Leu Lys Lys Leu Ile Gly Glu Arg Ser Gly Ser Pro Glu Asp Glu Ile 260 265 270 cag gca ctg gaa gaa gaa aat get caa ctg gaa cag gaa aac gcg gcg 864 Gin Al a Leu Glu Glu Glu Asn Ala Gin Leu Glu Gin Glu Asn Ala Ala 275 280 285 ctg gaa gaa gaa ate gca cag ctg gaa tac ggc tcc ggg gtt ccg ggt 912 Leu Glu Glu Glu Ile Ala Gin Leu Glu Tyr Gly Ser Gly Val Pro Gly 290 295 300 gtt ggt gtt cct ggc gtg ggt gtt cct ggt gtt ggc gtg cca ggt gtg 960 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 305 310 315 320 gga gtg cca ggc gtt ggt gta ccg ggt gtg ggc gtt ccg gga gtc gga 1008 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 325 330 335 gtt ccg gga gta ggc gtg cct ggt gtt gtt ccg ggt gta ggt tcc 1056 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Ser 340 345 350 gga act agt 1065
Gly Thr Ser 355 <210> 6 <211> 355 <212> PRT <213> Sintetični 6 <4 00> -41- ····Gly Thr Ser 355 < 210 > 6 < 211 > 355 < 212 > PRT < 213 > Synthetic 6 < 4 00 > -41- ····
Met His Thr Pro Glu His Ile Thr Ala Val Val Gin Arg Phe Val Ala 1 5 10 15 Al a Leu As n Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp 20 25 30 Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr 35 40 45 Al a Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala 50 55 60 Val Glu Leu Thr Gin Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe 65 70 75 80 Ala Phe Thr Val Ser Phe Glu Tyr Gin Gly Arg Lys Thr Val Val Ala 85 90 95 Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile 100 105 110 Ar g Ala Leu Phe Gly Glu Lys As n Ile His Ala Cys Gin Met Asp Pro 115 120 125 Met Tyr His His His His His His Ser Arg Ala Gly Leu Leu Gly Asp 130 135 140 Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu Phe Lys Arg Ile 145 150 155 160 Val Gin Ar g Ile Lys Asp Phe Leu Arg Asn Leu Val Pro Arg Thr Glu 165 170 175 Ser Ser Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 180 185 190 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 195 200 205 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 210 215 220 Val Pro Gly Val Gly Ser Gly Arg Met Lys Gin Leu Glu Asp Lys Ile 225 230 235 240 Glu Glu Leu Leu Ser Lys Ile Tyr His Leu Glu Asn Glu Ile Ala Arg 245 250 255 Leu Lys Lys Leu Ile Gly Glu Arg Ser Gly Ser Pro Glu Asp Glu Ile 260 265 270 Gin Ala Leu Glu Glu Glu As n Ala Gin Leu Glu Gin Glu Asn Ala Ala 275 280 285 Leu Glu Glu Glu Ile Ala Gin Leu Glu Tyr Gly Ser Gly Val Pro Gly 290 295 300 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 305 310 315 320Met His Thr Pro Glu His Ile Thr Ala Val Val Gin Arg Phe Val Ala 1 5 10 15 Al a Leu As n Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp 20 25 30 Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr 35 40 45 Al a Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala 50 55 60 Val Glu Leu Thr Gin Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe 65 70 75 80 Ala Phe Thr Val Ser Phe Glu Tyr Gin Gly Arg Lys Thr Val Val Ala 85 90 95 Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile 100 105 110 Ar g Ala Leu Phe Gly Glu Lys As n Ile His Ala Cys Gin Met Asp Pro 115 120 125 Met Tyr His His His His His Ser Arg Ala Gly Leu Leu Gly Asp 130 135 140 Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu Phe Lys Arg Ile 145 150 155 160 Val Gin Ar g Ile Lys Asp Phe Leu Arg Asn Leu Val Pro Arg Thr Glu 165 170 175 Ser Se r Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 180 185 190 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 195 200 205 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 210 215 220 Val Pro Gly Val Gly Ser Gly Arg Met Lys Gin Leu Glu Asp Lys Ile 225 230 235 240 Glu Glu Leu Leu Ser Lys Ile Tyr His Leu Glu Asn Glu Ile Ala Arg 245 250 255 Leu Lys Lys Leu Ile Gly Glu Arg Ser Gly Ser Pro Glu Asp Glu Ile 260 265 270 Gin Ala Leu Glu Glu Glu As n Ala Gin Leu Glu Gin Glu Asn Ala Ala 275 280 285 Leu Glu Glu Glu Ile Ala Gin Leu Glu Tyr Gly Ser Gly Val Pro Gly 290 295 300 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 305 310 315 320
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 325 330 335 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 340 345 350Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 325 330 335 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 340 345 350
Gly Thr Ser 355 <210> 7 <211> 1065 <212> DNK <213> Sintetični <220> <221> CDS <222> (1)..(1065) <223> KSI-DP-Histag-LL37-ELST-GCN- P2-ELST <400> 7 atg cat acc cca gaa cac ate acc gcc gtg gta cag cgc ttt gtg gct 48 Met His Thr Pro Glu His Ile Thr Ala Val Val Gin Arg Phe Val Ala 1 5 10 15 gcg ctc aat gcc ggc gat ctg gac ggc ate gtc gcg ctg ttt gcc gat 96 Al a Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp 20 25 30 gac gcc acg gtg gaa gac ccc gtg ggt tcc gag ccc agg tcc ggt acg 144 Asp Al a Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr 35 40 45 gct gcg att cgt gag ttt tac gcc aac teg ctc aaa ctg cct ttg gcg 192 Al a Al a Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala 50 55 60 gtg gag ctg acg cag gag gta cgc gcg gtc gcc aac gaa gcg gcc ttc 240 Val Glu Leu Thr Gin Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe 65 70 75 80 gct ttc acc gtc agc ttc gag tat cag ggc cgc aag acc gta gtt gcg 288 Al a Phe Thr Val Ser Phe Glu Tyr Gin Gly Arg Lys Thr Val Val Ala 85 90 95 ccc ate gat cac ttt cgc ttc aat ggc gcc ggc aag gtg gtg agc ate 336 Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile 100 105 110 cgc gcc ttg ttt ggc gag aag aat att cac gca tgc cag atg gat cct 384 Ar g Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gin Met Asp Pro 115 120 125 atg tat cat cac cat cac cat cac tet aga gcc ggc ctg ctg ggt gat 432 Met Tyr His His His His His His Ser Arg Ala Gly Leu Leu Gly Asp 130 135 140 ttc ttc cgg aaa tet aaa gag aag att ggc aaa gag ttt aaa aga att 480 Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu Phe Lys Arg Ile 145 150 155 160 gtc cag aga ate aag gat ttt ttg cgg aat ctt gta ccc agg aca gag 528 -43-Gly Thr Ser 355 < 210 > 7 < 211 > 1065 < 212 > DNA < 213 > Synthetic < 220 > < 221 > CDS < 222 > (1) .. (1065) < 223 > KSI-DP-Histag-LL37-ELST-GCN-P2-ELST < 400 > 7 atg cat acc cca gaa cac ate acc gcc gtg gta cag cgc ttt gtg gct 48 Met His Thr Pro Glu His Ile Thr Ala Val Val Gin Arg Phe Val Ala 1 5 10 15 gcg ctc aat gcc ggc gat ctg gac ggc ate gtc gcg ctg ttt gcc gat 96 Al a Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp 20 25 30 gac gcc acg gtg gaa gac ccc gtg ggt tcc gag ccc agg tcc ggt acg 144 Asp Al a Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr 35 40 45 gct gcg att cgt gag ttt tac gcc aac teg ctc aaa ctg cct ttg gcg 192 Al a Al a Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala 50 55 60 gtg gag ctg acg cag gag gta cgc gcg gtc gcc aac gaa gcg gcc ttc 240 Val Glu Leu Thr Gin Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe 65 70 75 80 gct ttc acc gtc agc ttc gag tat cag ggc cgc aag acc gta gtt gcg 288 Al a Phe Thr Val Ser Phe Glu Tyr Gin Gly Arg Lys Thr Val Val Ala 85 90 95 ccc ate gat cac ttt cgc ttc aat ggc gcc ggc aag gtg gtg agc ate 336 Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile 100 105 110 cgc gcc ttg ttt ggc gag aag aat att cac gca tgc cag atg gat cct 384 Ar g Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gin Met Asp Pro 115 120 125 atg tat cat cac cat cac cat cac tet aga gcc ggc ctg ctg ggt gat 432 Met Tyr His His His His His Ser Arg Ala Gly Leu Leu Gly Asp 130 135 140 ttc ttc cgg aaa tet aaa gag aag att ggc aaa gag ttt aaa aga att 480 Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu Phe Lys Arg Ile 145 150 155 160 gtc cag aga ate aag gat ttt ttg cgg aat ctt gta aca gag 528 -43-
Val Gin Ar g Ile Lys Asp Phe Leu Arg 165 tcc tcc ggg gtt ccg ggt gtt ggt gtt Ser Ser Gly Val Pro Gly Val Gly Val 180 185 gtt ggc gtg cca ggt gtg gga gtg cca Val Gly Val Pro Gly Val Gly Val Pro 195 200 ggc gtt ccg gga gtc gga gtt ccg gga Gly Val Pro Gly Val Gly Val Pro Gly 210 215 gtt ccg ggt gta ggt tcc ggg cgt atg Val Pro Gly Val Gly Ser Gly Arg Met 225 230 gag gag ctg ctg tet aag ate tac cac Glu Glu Leu Leu Ser Lys Ile Tyr His 245 ctg aaa aag ctg att ggt gaa ege tcc Leu Lys Lys Leu Ile Gly Glu Arg Ser 260 265 gca cag ctg aaa gaa aag aac gcg gcc Ala Gin Leu Lys Glu Lys Asn Ala Ala 275 280 ctg aag gag aaa ate caa gca ctg aaa Leu Lys Glu Lys Ile Gin Ala Leu Lys 290 295 gtt ggt gtt cct ggc gtg ggt gtt cct Val Gly Val Pro Gly Val Gly Val Pro 305 310 gga gtg cca ggc gtt ggt gta ccg ggt Gly Val Pro Gly Val Gly Val Pro Gly 325 gtt ccg gga gta ggc gtg cct ggt gtt Val Pro Gly Val Gly Val Pro Gly Val 340 345 gga act agt Gly Thr Ser 355 <210> 8 <211> 355 <212> PRT <213> Sintetični <4 00> 8 Met His Thr Pro Glu His Ile Thr Ala 1 5Val Gin Ar g Ile Lys Asp Phe Leu Arg 165 tcc tcc ggg gtt ccg ggt gtt ggt gtt Ser Ser Gly Val Pro Gly Val Gly Val 180 185 gtt ggc gtg cca ggt gtg gga gtg cca Val Gly Val Pro Gly Val Gly Val Pro 195 200 ggc gtt ccg gga gtc gga gtt ccg gga Gly Val Pro Gly Val Gly Val Pro Gly 210 215 gtt ccg ggt gta ggt tcc ggg cgt atg Val Pro Gly Val Gly Ser Gly Arg Met 225 230 gag gag ctg ctg tet aag ate tac cac Glu Glu Leu Leu Ser Lys Ile Tyr His 245 ctg aaa aag ctg att ggt gaa ege tcc Leu Lys Lys Leu Ile Gly Glu Arg Ser 260 265 gca cag ctg aaa gaa aag aac gcg gcc Ala Gin Leu Lys Glu Lys Asn Ala Ala5 ctg aag gag aaa ate caa gca ctg aaa Leu Lys Glu Lys Ile Gin Ala Leu Lys 290 295 gtt ggt gtt cct ggc gtg ggt gtt cct Val Gly Val Pro Gly Val Gly Val Pro 305 310 gga gtg cca ggc gtt ggt gta cc Val Pro Gly Val Gly Val Pro Gly 325 gtt ccg gga gta ggc gtg cct ggt gtt Val Pro Gly Val Gl y Val Pro Gly Val 340 345 gga act agt Gly Thr Ser 355 < 210 > 8 < 211 > 355 < 212 > PRT < 213 > Synthetic < 4 00 > 8 Met His Thr Pro Glu His Ile Thr Ala 1 5
Asn Leu Val Pro Arg Thr Glu 170 175 cct ggc gtg ggt gtt cct ggt 576 Pro Gly Val Gly Val Pro Gly 190 ggc gtt ggt gta ccg ggt gtg 624 Gly Val Gly Val Pro Gly Val 205 gta ggc gtg cct ggt gtt ggg 672 Val Gly Val Pro Gly Val Gly 220 aaa cag ctg gaa gat aaa ate 720 Lys Gin Leu Glu Asp Lys Ile 235 240 ctg gaa aac gaa att get ege 768 Leu Glu Asn Glu Ile Ala Arg 250 255 ggg tet cca gaa gac aaa ate 816 Gly Ser Pro Glu Asp Lys Ile 270 ctg aaa gaa aag aat caa cag 864 Leu Lys Glu Lys Asn Gin Gin 285 tat ggc tcc ggg gtt ccg ggt 912 Tyr Gly Ser Gly Val Pro Gly 300 ggt gtt ggc gtg cca ggt gtg 960 Gly Val Gly Val Pro Gly Val 315 320 gtg ggc gtt ccg gga gtc gga 1008 Val Gly Val Pro Gly Val Gly 330 335 ggg gtt ccg ggt gta ggt tcc 1056 Gly Val Pro Gly Val Gly Ser 350 1065Asn Leu Val Pro Arg Thr Glu 170 175 cct ggc gtg ggt gtt cct ggt 576 Pro Gly Val Gly Val Pro Gly 190 ggc gtt ggt gta ccg ggt gtg 624 Gly Val Gly Val Pro Gly Val 205 gta ggc gtg cct ggt gtt ggg 672 Val Gly Val Pro Gly Val Gly 220 aaa cag ctg gaa gat aaa ate 720 Lys Gin Leu Glu Asp Lys Ile 235 240 ctg gaa aac gaa att get ege 768 Leu Glu Asn Glu Ile Ala Arg 250 255 ggg tet cca gaa gac aaa ate 816 Gly Ser Pro Glu Asp Lys Ile 270 ctg aaa gaa aag aat caa cag 864 Leu Lys Glu Lys Asn Gin Gin 285 tat ggc tcc ggg gtt ccg ggt 912 Tyr Gly Ser Gly Val Pro Gly 300 ggt gtt ggc gtg cca ggt gtg 960 Gly Val Pro Gly Val 315 320 gtg ggc gtt ccg gga gtc gga 1008 Val Gly Val Pro Gly Val Gly 330 335 ggg gtt ccg ggt gta ggt tcc 1056 Gly Val Pro Gly Val Gly Ser 350 1065
Val Val Gin Arg Phe Val Ala 10 15 -44- » ····Val Val Gin Arg Phe Val Ala 10 15 -44- »····
Al a Leu As n Al a Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp 20 25 30 Asp Al a Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr 35 40 45 Al a Al a Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala 50 55 60 Val Glu Leu Thr Gin Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe 65 70 75 80 Al a Phe Thr Val Ser Phe Glu Tyr Gin Gly Arg Lys Thr Val Val Ala 85 90 95 Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile 100 105 110 Arg Al a Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gin Met Asp Pro 115 120 125 Met Tyr His His His His His His Ser Arg Ala Gly Leu Leu Gly Asp 130 135 140 Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu Phe Lys Arg Ile 145 150 155 160 Val Gin Arg Ile Lys Asp Phe Leu Arg Asn Leu Val Pro Arg Thr Glu 165 170 175 Ser Ser Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 180 185 190 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 195 200 205 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 210 215 220 Val Pro Gly Val Gly Ser Gly Arg Met Lys Gin Leu Glu Asp Lys Ile 225 230 235 240 Glu Glu Leu Leu Ser Lys Ile Tyr His Leu Glu Asn Glu Ile Ala Arg 245 250 255 Leu Lys Lys Leu Ile Gly Glu Arg Ser Gly Ser Pro Glu Asp Lys Ile 260 265 270 Al a Gin Leu Lys Glu Lys Asn Ala Ala Leu Lys Glu Lys Asn Gin Gin 275 280 285 Leu Lys Glu Lys Ile Gin Ala Leu Lys Tyr Gly Ser Gly Val Pro Gly 290 295 300 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 305 310 315 320 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 325 330 335Al a Leu As n Al a Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp 20 25 30 Asp Al a Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr 35 40 45 Al a Al a Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala 50 55 60 Val Glu Leu Thr Gin Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe 65 70 75 80 Al a Phe Thr Val Ser Phe Glu Tyr Gin Gly Arg Lys Thr Val Val Ala 85 90 95 Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile 100 105 110 Arg Al a Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gin Met Asp Pro 115 120 125 Met Tyr His His His His His His Ser Arg Ala Gly Leu Leu Gly Asp 130 135 140 Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu Phe Lys Arg Ile 145 150 155 160 Val Gin Arg Ile Lys Asp Phe Leu Arg Asn Leu Val Pro Arg Thr Glu 165 170 175 Ser Ser Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 180 185 190 V al Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 195 200 205 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 210 215 220 Val Pro Gly Val Gly Ser Gly Arg Met Lys Gin Leu Glu Asp Lys Ile 225 230 235 240 Glu Glu Leu Leu Ser Lys Ile Tyr His Leu Glu Asn Glu Ile Ala Arg 245 250 255 Leu Lys Lys Leu Ile Gly Glu Arg Ser Gly Ser Pro Glu Asp Lys Ile 260 265 270 Al a Gin Leu Lys Glu Lys Asn Ala Ala Leu Lys Glu Lys Asn Gin Gin 275 280 285 Leu Lys Glu Lys Ile Gin Ala Leu Lys Tyr Gly Ser Gly Val Pro Gly 290 295 300 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 305 310 315 320 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 325 330 335
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Ser • ••· • ·1 -45- » 340 345 350Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Ser • •• · • · 1 -45- »340 345 350
Gly Thr Ser 355 <210> 9 <211> 99 <212> DNK <213> Sintetični <220> <221> CDS <222> (1)..(99) <223> Pl <4 00> 9 agc cca gaa gac gaa att Ser Pro Glu Asp Glu Ile 1 5 gaa cag gaa aac gcg gcg Glu Gin Glu Asn Ala Ala 20 ggc Gly cag gca ctg gaa gaa gaa Gin Ala Leu Glu Glu Glu 10 ctg gaa gaa gaa ate gca Leu Glu Glu Glu Ile Ala 25 aat get caa ctg 48 Asn Ala Gin Leu 15 cag ctg gaa tac 96 Gin Leu Glu Tyr 30 99 <210> 10 <211> 33 <212> PRT <213> Sintetični <400> 10 Ser Pro Glu Asp Glu Ile 1 5 Glu Gin Glu Asn Ala Ala 20 GlyGly Thr Ser 355 < 210 > 9 < 211 > 99 < 212 > DNA < 213 > Synthetic < 220 > < 221 > CDS < 222 > (1) .. (99) < 223 > Pl < 4 00 > 9 agc cca gaa gac gaa att Ser Pro Glu Asp Glu Ile 1 5 gaa cag gaa aac gcg gcg Glu Gin Glu Asn Ala Ala 20 ggc Gly cag gca ctg gaa gaa gaa Gin Ala Leu Glu Glu Glu 10 ctg gaa gaa gaa ate gca Leu Glu Glu Glu Ile Ala 25 aat get caa ctg 48 Asn Ala Gin Leu 15 cag ctg gaa tac 96 Gin Leu Glu Tyr 30 99 < 210 > 10 < 211 > 33 < 212 > PRT < 213 > Synthetic < 400 > 10 Ser Pro Glu Asp Glu Ile 1 5 Glu Gin Glu Asn Ala Ala 20 Gly
Gin Ala Leu Glu Glu Glu 10 Leu Glu Glu Glu Ile Ala 25Gin Ala Leu Glu Glu Glu 10 Leu Glu Glu Glu Ile Ala 25
Asn Ala Gin Leu 15 Gin Leu Glu Tyr 30 <210> 11 <211> 99 <212> DNK <213> Sintetični <220> <221> CDS <222> (D ·. • (99) <223> P2 <4 00> 11 tet cca gaa gac aaa ate Ser Pro Glu Asp Lys Ile 1 5 aaa gaa aag aat caa cag Lys Glu Lys Asn Gin Gin gca cag ctg aaa gaa aag Ala Gin Leu Lys Glu Lys 10 ctg aag gag aaa ate caa Leu Lys Glu Lys Ile Gin aac gcg gcc ctg 48Asn Ala Gin Leu 15 Gin Leu Glu Tyr 30 < 210 > 11 < 211 > 99 < 212 > DNA < 213 > Synthetic < 220 > < 221 > CDS < 222 > (D ·. • (99) < 223 > P2 < 4 00 > 11 tet cca gaa gac aaa ate Ser Pro Glu Asp Lys Ile 1 5 aaa gaa aag aat caa cag Lys Glu Lys Asn Gin Gin gca cag ctg aaa gaa aag Ala Gin Leu Lys Glu Lys 10 ctg aag gag aaa ate caa Leu Lys Glu Lys Ile Gin aac gcg gcc ctg 48
Asn Ala Ala Leu 15 gca ctg aaa tat 96Asn Ala Ala Leu 15 gca ctg aaa tat 96
Ala Leu Lys Tyr • · -46- • · · ggc Gly 20 <210> 12 <211> 33 <212> PRT <213> Sintetični <400> 12Ala Leu Lys Tyr • · -46- • · · ggc Gly 20 < 210 > 12 < 211 > 33 < 212 > PRT < 213 > Synthetic < 400 > 12
Ser Pro Glu Asp Lys Ile Ala Gin Leu 1 5 Lys Glu Lys Asn Gin Gin Leu Lys Glu 20 25 GlySer Pro Glu Asp Lys Ile Ala Gin Leu 1 5 Lys Glu Lys Asn Gin Gin Leu Lys Glu 20 25 Gly
Lys Glu Lys Asn Ala Ala Leu 10 15 Lys Ile Gin Ala Leu Lys Tyr 30 <210> 13 <211> 99 <212> DNK <213> Sintetični <220> <221> CDS <222> (1)..(99) <223> GCN <4 00> 13 cgt atg aaa cag ctg gaa gat aaa ate Arg Met Lys Gin Leu Glu Asp Lys Ile 1 5 tac cac ctg gaa aac gaa att get ege Tyr His Leu Glu Asn Glu Ile Ala Arg 20 25 ege Arg gag gag ctg ctg tet aag ate 48 Glu Glu Leu Leu Ser Lys Ile 10 15 ctg aaa aag ctg att ggt gaa 96 Leu Lys Lys Leu Ile Gly Glu 30 99 <210> 14 <211> 33 <212> PRT <213> Sintetični <400> 14 Arg Met Lys Gin Leu Glu Asp Lys Ile 1 5 Tyr His Leu Glu Asn Glu Ile Ala Arg 20 25Lys Glu Lys Asn Ala Ala Leu 10 15 Lys Ile Gin Ala Leu Lys Tyr 30 < 210 > 13 < 211 > 99 < 212 > DNA < 213 > Synthetic < 220 > < 221 > CDS < 222 > (1) .. (99) < 223 > GCN < 4 00 > 13 cgt atg aaa cag ctg gaa gat aaa ate Arg Met Lys Gin Leu Glu Asp Lys Ile 1 5 tac cac ctg gaa aac gaa att get ege Tyr His Leu Glu Asn Glu Ile Ala Arg 20 25 ege Arg gag gag ctg ctg tet aag ate 48 Glu Glu Leu Leu Ser Lys Ile 10 15 ctg aaa aag ctg att ggt gaa 96 Leu Lys Lys Leu Ile Gly Glu 30 99 < 210 > 14 < 211 > 33 < 212 > PRT < 213 > Synthetic < 400 > 14 Arg Met Lys Gin Leu Glu Asp Lys Ile 1 5 Tyr His Leu Glu Asn Glu Ile Ala Arg 20 25
Glu Glu Leu Leu Ser Lys Ile 10 15 Leu Lys Lys Leu Ile Gly Glu 30Glu Glu Leu Leu Ser Lys Ile 10 15 Leu Lys Lys Leu Ile Gly Glu 30
Arg -47-Arg -47-
<210 > 15 <211> 111 <212> DNK <213> Sintetični <220> <221> CDS <222> (1)..(111) <223> LL37 <4 00> 15 ctg ctg ggt gat ttc ttc cgg aaa tet aaa gag aag att ggc aaa gag Leu Leu Gly Asp Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu 1 5 10 15 ttt aaa aga att gtc cag aga ate aag gat ttt ttg cgg aat ctt gta Phe Lys Arg Ile Val Gin Arg Ile Lys Asp Phe Leu Arg Asn Leu Val 20 25 30 ccc agg aca gag tcc Pro Arg Thr Glu Ser 35 <210> 16 <211> 37 <212> PRT <213> Sintetični <400> 16 48 96 111< 210 > 15 < 211 > 111 < 212 > DNA < 213 > Synthetic < 220 > < 221 > CDS < 222 > (1) .. (111) < 223 > LL37 < 4 00 > 15 ctg ctg ggt gat ttc ttc cgg aaa tet aaa gag aag att ggc aaa gag Leu Leu Gly Asp Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu 1 5 10 15 ttt aaa aga att gtc cag aga ate aag cat aat ctt gta Phe Lys Arg Ile Val Gin Arg Ile Lys Asp Phe Leu Arg Asn Leu Val 20 25 30 ccc agg aca gag tcc Pro Arg Thr Glu Ser 35 < 210 > 16 < 211 > 37 < 212 > PRT < 213 > Synthetic < 400 > 16 48 96 111
Leu Leu Gly Asp Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu 1 5 10 15 Phe Lys Arg Ile Val Gin Arg Ile Lys Asp Phe Leu Arg Asn Leu Val 20 25 30 Pro Arg Thr Glu Ser 35 <210> 17 <211> 150 <212> DNK <213> Sintetični <220> <221> CDS <222> (D . . (150) <223> ELST <400> 17 gtt ccg ggt gtt ggt gtt cct ggc gtg ggt gtt cct ggt gtt ggc gtg Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 1 5 10 15 cca ggt gtg gga gtg cca ggc gtt ggt gta ccg ggt gtg ggc gtt ccg Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 20 25 30 gga gtc gga gtt ccg gga gta ggc gtg cct ggt gtt ggg gtt ccg ggt Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 35 40 45 48 96 144 -48- ··· gta ggt 150Leu Leu Gly Asp Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu 1 5 10 15 Phe Lys Arg Ile Val Gin Arg Ile Lys Asp Phe Leu Arg Asn Leu Val 20 25 30 Pro Arg Thr Glu Ser 35 < 210 > 17 < 211 > 150 < 212 > DNA < 213 > Synthetic < 220 > < 221 > CDS < 222 > (D.. (150) < 223 > ELST < 400 > 17 gtt ccg ggt gtt ggt gtt cct ggc gtg ggt gtt cct ggt gtt ggc gtg Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 1 5 10 15 cca ggt gtg gga gtg cca ggc gtt ggt gta ccg ggt gtg ggc gtt ccg Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 20 25 30 gga gtc gga gtt ccg gga gta ggc gtg cct ggt gtt ggg gtt ccg ggt Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 35 40 45 48 96 144 -48- ··· gta ggt 150
Val Gly 50 <210> 18 <211> 50 <212> PRT <213> Sintetični <4 00> 18Val Gly 50 < 210 > 18 < 211 > 50 < 212 > PRT < 213 > Synthetic < 4 00 > 18
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 1 5 10 15 Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 20 25 30 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 35 40 45 Val Gly 50 <210> 19 <211> 1140 <212> DNK <213> Sintetični <220> <221> CDS <222> (D · .(1140) <223> KSI-I DP-Histag-ELST-GCN- : P2-ELST-AEA <4 00> 19 atg cat acc cca gaa cac ate acc gcc gtg gta cag ege ttt gtg gct 48 Met His Thr Pro Glu His Ile Thr Ala Val Val Gin Arg Phe Val Ala 1 5 10 15 gcg ctc aat gcc ggc gat ctg gac ggc ate gtc gcg ctg ttt gcc gat 96 Ala Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp 20 25 30 gac gcc acg gtg gaa gac ccc gtg ggt tcc gag ccc agg tcc ggt acg 144 As p Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr 35 40 45 gct gcg att cgt gag ttt tac gcc aac teg ctc aaa ctg cct ttg gcg 192 Ala Ala Ile Ar g Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala 50 55 60 gtg gag ctg acg cag gag gta ege gcg gtc gcc aac gaa gcg gcc ttc 240 Val Glu Leu Thr Gin Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe 65 70 75 80 gct ttc acc gtc agc ttc gag tat cag ggc ege aag acc gta gtt gcg 288 Ala Phe Thr Val Ser Phe Glu Tyr Gin Gly Arg Lys Thr Val Val Ala 85 90 95 ccc ate gat cac ttt ege ttc aat ggc gcc ggc aag gtg gtg agc ate 336 Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile -49- 100 105 110 cgc gcc ttg ttt ggc gag aag aat att cac gca tgc cag atg gat cct 384 Arg Ala Leu Phe Gly Glu Lys As n Ile His Ala Cys Gin Met Asp Pro 115 120 125 atg tat cat cac cat cac cat cac tet aga gcc ggc gtt ccg ggt gtt 432 Met Tyr His His His His His His Ser Arg Ala Gly Val Pro Gly Val 130 135 140 ggt gtt cct ggc gtg ggt gtt cct ggt gtt ggc gtg cca ggt gtg gga 480 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 145 150 155 160 gtg cca ggc gtt ggt gta ccg ggt gtg ggc gtt ccg gga gtc gga gtt 528 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 165 170 175 ccg gga gta ggc gtg cct ggt gtt ggg gtt ccg ggt gta ggt tcc ggg 576 Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Ser Gly 180 185 190 cgt atg aaa cag ctg gaa gat aaa ate gag gag ctg ctg tet aag ate 624 Arg Met Lys Gin Leu Glu Asp Lys Ile Glu Glu Leu Leu Ser Lys Ile 195 200 205 tac cac ctg gaa aac gaa att get cgc ctg aaa aag ctg att ggt gaa 672 Tyr His Leu Glu As n Glu Ile Ala Arg Leu Lys Lys Leu Ile Gly Glu 210 215 220 cgc tcc ggg tet cca gaa gac aaa ate gca cag ctg aaa gaa aag aac 720 Arg Ser Gly Ser Pro Glu Asp Lys Ile Ala Gin Leu Lys Glu Lys Asn 225 230 235 240 gcg gcc ctg aaa gaa aag aat caa cag ctg aag gag aaa ate caa gca 768 Al a Ala Leu Lys Glu Lys As n Gin Gin Leu Lys Glu Lys Ile Gin Ala 245 250 255 ctg aaa tat ggc tcc ggg gtt ccg ggt gtt ggt gtt cct ggc gtg ggt 816 Leu Lys Tyr Gly Ser Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 260 265 270 gtt cct ggt gtt ggc gtg cca ggt gtg gga gtg cca ggc gtt ggt gta 864 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 275 280 285 ccg ggt gtg ggc gtt ccg gga gtc gga gtt ccg gga gta ggc gtg cct 912 Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 290 295 300 ggt gtt ggg gtt ccg ggt gta ggt tcc ggg aag tgg get gcc ate gaa 960 Gly Val Gly Val Pro Gly Val Gly Ser Gly Lys Trp Ala Ala Ile Glu 305 310 315 320 gaa gaa gca gcc get att aaa gaa gag gca gca gcg ate gaa gaa aaa 1008 Glu Glu Ala Ala Ala Ile Lys Glu Glu Ala Ala Ala Ile Glu Glu Lys 325 330 335 gcc gca gcc ate aaa gag gaa gcc gca get ate gag gaa aaa tgg get 1056 Ala Ala Ala Ile Lys Glu Glu Ala Ala Ala Ile Glu Glu Lys Trp Ala 340 345 350 • · -50- gcg att gaa gag gaa gct gcg gca ata gaa gag aaa tgg gct gct ate Ala Ile Glu Glu Glu Ala Ala Ala Ile Glu Glu Lys Trp Ala Ala Ile 355 360 365 aaa gaa aaa gcg gct gca ate aaa tcc gga act agt Lys Glu Lys Ala Ala Ala Ile Lys Ser Gly Thr Ser 370 375 380 <210> 20 <211> 380 <212> PRT <213> Sintetični <400> 20 Met His Thr Pro Glu His Ile Thr Ala Val Val Gin Arg Phe Val Ala 1 5 10 15 Ala Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp 20 25 30 Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr 35 40 45 Ala Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala 50 55 60 Val Glu Leu Thr Gin Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe 65 70 75 80 Ala Phe Thr Val Ser Phe Glu Tyr Gin Gly Arg Lys Thr Val Val Ala 85 90 95 Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile 100 105 110 Arg Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gin Met Asp Pro 115 120 125 Met Tyr His His His His His His Ser Arg Ala Gly Val Pro Gly Val 130 135 140 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 145 150 155 160 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 165 170 175 Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Ser Gly 180 185 190 Arg Met Lys Gin Leu Glu Asp Lys Ile Glu Glu Leu Leu Ser Lys Ile 195 200 205 Tyr His Leu Glu Asn Glu Ile Ala Arg Leu Lys Lys Leu Ile Gly Glu 210 215 220 Arg Ser Gly Ser Pro Glu Asp Lys Ile Ala Gin Leu Lys Glu Lys Asn 225 230 235 240 1104 1140Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 1 5 10 15 Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 20 25 30 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 35 40 45 Val Gly 50 < 210 > 19 < 211 > 1140 < 212 > DNA < 213 > Synthetic < 220 > < 221 > CDS < 222 > (D ·. (1140) < 223 > KSI-I DP-Histag-ELST-GCN-: P2-ELST-AEA < 4 00 > 19 atg cat acc cca gaa cac ate acc gcc gtg gta cag ege ttt gtg gct 48 Met His Thr Pro Glu His Ile Thr Ala Val Val Gin Arg Phe Val Ala 1 5 10 15 gcg ctc aat gcc ggc gat ctg gac ggc ate gtc gcg ctg ttt gcc gat 96 Ala Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp 20 25 30 gac gcc acg gtg gaa gac ccc gtg ggt tcc gag ccc agg tcc ggt acg 144 As p Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr 35 40 45 gct gcg att cgt gag ttt tac gcc aac teg ctc aaa ctg cct ttg gcg 192 Ala Ala Ile Ar g Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala 50 55 60 gtg gag ctg acg cag gag gta ege gcg gtc gcc aac gaa gcg gcc ttc 240 Val Glu Leu Thr Gin Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe 65 70 75 80 gct ttc acc gtc agc ttc gag tat cag ggc ege aag acc gta gtt gcg 288 Ala Phe Thr Val Se r Phe Glu Tyr Gin Gly Arg Lys Thr Val Val Ala 85 90 95 ccc ate gat cac ttt ege ttc aat ggc gcc ggc aag gtg gtg agc ate 336 Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile -49 - 100 105 110 cgc gcc ttg ttt ggc gag aag aat att cac gca tgc cag atg gat cct 384 Arg Ala Leu Phe Gly Glu Lys As n Ile His Ala Cys Gin Met Asp Pro 115 120 125 atg tat cat cac cat cac cat cac tet aga gcc ggc gtt ccg ggt gtt 432 Met Tyr His His His His His His Ser Arg Ala Gly Val Pro Gly Val 130 135 140 ggt gtt cct ggc gtg ggt gtt cct ggt gtt ggc gtg cca ggt gtg gga 480 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 145 150 155 160 gtg cca ggc gtt ggt gta ccg ggt gtg ggc gtt ccg gga gtc gga gtt 528 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 165 170 175 ccg gga gta ggc gtg cct ggt gtt ggg gtt ccg ggt gta ggt tcc ggg 576 Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly S er Gly 180 185 190 cgt atg aaa cag ctg gaa gat aaa ate gag gag ctg ctg tet aag ate 624 Arg Met Lys Gin Leu Glu Asp Lys Ile Glu Glu Leu Leu Ser Lys Ile 195 200 205 tac cac ctg gaa aac gaa att get cgc ctg aaa aag ctg att ggt gaa 672 Tyr His Leu Glu As n Glu Ile Ala Arg Leu Lys Lys Leu Ile Gly Glu 210 215 220 cgc tcc ggg tet cca gaa gac aaa ate gca cag ctg aaa gaa aag aac 720 Arg Ser Gly Ser Pro Glu Asp Lys Ile Ala Gin Leu Lys Glu Lys Asn 225 230 235 240 gcg gcc ctg aaa gaa aag aat caa cag ctg aag gag aaa ate caa gca 768 Al a Ala Leu Lys Glu Lys As n Gin Gin Leu Lys Glu Lys Ile Gin Ala 245 250 255 ctg aaa tat ggc tcc ggg gtt ccg ggt gtt ggt gtt cct ggc gtg ggt 816 Leu Lys Tyr Gly Ser Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 260 265 270 gtt cct ggt gtt ggc gtg cca ggt gtg cca ggc gtt ggt gta 864 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 275 280 285 ccg ggt gtg ggc gtt ccg gga gtc gga gtt ccg gga gta ggc gtg cct 912 Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 290 295 300 ggt gtt ggg gtt ccg ggt gta ggt tgg ggg a gcc ate gaa 960 Gly Val Gly Val Pro Gly Val Gly Ser Gly Lys Trp Ala Ala Ile Glu 305 310 315 320 gaa gaa gca gcc get att aaa gaa gag gca gca gcg ate gaa gaa aaa 1008 Glu Glu Ala Ala Ala Ile Lys Glu Glu Ala Ala Ala Ile Glu Glu Lys 325 330 335 gcc gca gcc ate aaa gag gaa gcc gca get ate gag gaa aaa tgg get 1056 Ala Ala Ala Ile Lys Glu Glu Ala Ala Ala Ile Glu Glu Lys Trp Ala 340 345 350 • · -50 - gcg att gaa gag gaa gct gcg gca ata gaa gag aaa tgg gct gct ate Ala Ile Glu Glu Glu Ala Ala Ala Ile Glu Glu Lys Trp Ala Ala Ile 355 360 365 aaa gaa aaa gcg gct gca ate aaa tcc gga act agt Lys Glu Lys Ala Ala Ala Ile Lys Ser Gly Thr Ser 370 375 380 < 210 > 20 < 211 > 380 < 212 > PRT < 213 > Synthetic < 400 > 20 Met His Thr Pro Glu His Ile Thr Ala Val Val Gin Arg Phe Val Ala 1 5 10 15 Ala Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp 20 25 30 Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr 35 40 45 Ala Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala 50 55 60 Val Glu Leu Thr Gin Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe 65 70 75 80 Ala Phe Thr Val Ser Phe Glu Tyr Gin Gly Arg Lys Thr Val Val Ala 85 90 95 Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile 100 105 110 Arg Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gin Met Asp Pro 115 120 125 Met Tyr His His His His His Ser Arg Ala Gly Val Pro Gly Val 130 135 140 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 145 150 155 160 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 165 170 17 5 Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Ser Gly 180 185 190 Arg Met Lys Gin Leu Glu Asp Lys Ile Glu Glu Leu Leu Ser Lys Ile 195 200 205 Tyr His Leu Glu Asn Glu Ile Ala Arg Leu Lys Lys Leu Ile Gly Glu 210 215 220 Arg Ser Gly Ser Pro Glu Asp Lys Ile Ala Gin Leu Lys Glu Lys Asn 225 230 235 240 1104 1140
Ala Ala Leu Lys Glu Lys Asn Gin Gin 245 Leu Lys Tyr Gly Ser Gly Val Pro Gly 260 265 Val Pro Gly Val Gly Val Pro Gly Val 275 280 Pro Gly Val Gly Val Pro Gly Val Gly 290 295 Gly Val Gly Val Pro Gly Val Gly Ser 305 310 Glu Glu Ala Ala Ala Ile Lys Glu Glu 325 Ala Ala Ala Ile Lys Glu Glu Ala Ala 340 345 Ala Ile Glu Glu Glu Ala Ala Ala Ile 355 360 Lys Glu Lys Ala Ala Ala Ile Lys Ser 370 375 <210> 21 <211> 375 <212> DNK <213> Sintetični <220> <221> i CDS <222> (D · . . (375) <223> KSI <400> 21 cat acc cca gaa cac ate acc gcc gtg His Thr Pro Glu His Ile Thr Ala Val 1 5 ctc aat gcc ggc gat ctg gac ggc ate Leu Asn Ala Gly Asp Leu Asp Gly Ile 20 25 gcc acg gtg gaa gac ccc gtg ggt tcc Ala Thr Val Glu Asp Pro Val Gly Ser 35 40 gcg att cgt gag ttt tac gcc aac teg Ala Ile Arg Glu Phe Tyr Ala Asn Ser 50 55 gag ctg acg cag gag gta ege gcg gtc Glu Leu Thr Gin Glu Val Arg Ala Val 65 70 ttc acc gtc agc ttc gag tat cag ggc Phe Thr Val Ser Phe Glu Tyr Gin Gly 85Ala Ala Leu Lys Glu Lys Asn Gin Gin 245 Leu Lys Tyr Gly Ser Gly Val Pro Gly 260 265 Val Pro Gly Val Gly Val Pro Gly Val 275 280 Pro Gly Val Gly Val Pro Gly Val Gly 290 295 Gly Val Gly Val Pro Gly Val Gly Ser 305 310 Glu Glu Ala Ala Ala Ile Lys Glu Glu 325 Ala Ala Ala Ile Lys Glu Glu Ala Ala 340 345 Ala Ile Glu Glu Glu Ala Ala Ala Ile 355 360 Lys Glu Lys Ala Ala Ala Ile Lys Ser 370 375 < 210 > 21 < 211 > 375 < 212 > DNA < 213 > Synthetic < 220 > < 221 > and CDS < 222 > (D ·.. (375) < 223 > KSI < 400 > 21 cat acc cca gaa cac ate acc gcc gtg His Thr Pro Glu His Ile Thr Ala Val 1 5 ctc aat gcc ggc gat ctg gac ggc ate Leu Asn Ala Gly Asp Leu Asp Gly Ile 20 25 gcc acg gtg gaa gac ccc gtg ggt tcc Ala Thr Val Glu Asp Pro Val Gly Ser 35 40 gcg att cgt gag ttt tac gcc aac teg Ala Ile Arg Glu Phe Tyr Ala Asn Ser 50 55 gag ctg acg cag gag gta ege gcg gtc Glu Leu Thr Gin Glu Val Arg Ala Val 65 70 ttc acc gtc agc ttc gag tat cag ggc Phe Thr Val Ser Phe Glu Tyr Gin Gly 85
Leu 250 Lys Glu Lys Ile Gin 255 Ala Val Gly Val Pro Gly 270 Val Gly Gly Val Pro Gly 285 Val Gly Val Val Pro Gly 300 Val Gly Val Pro Gly Lys 315 Trp Ala Ala Ile Glu 320 Ala 330 Ala Ala Ile Glu Glu 335 Lys Ala Ile Glu Glu Lys 350 Trp Ala Glu Glu Lys Trp 365 Ala Ala Ile Gly Thr Ser 380 gta cag ege ttt gtg get gcg 48 Val 10 Gin Arg Phe Val Ala 15 Ala gtc gcg ctg ttt gcc gat gac 96 Val Ala Leu Phe Ala 30 Asp Asp gag ccc agg tcc ggt acg get 144 Glu Pro Arg Ser 45 Gly Thr Ala ctc aaa ctg cct ttg gcg gtg 192 Leu Lys Leu 60 Pro Leu Ala Val gcc aac gaa gcg gcc ttc get 240 Ala Asn 75 Glu Ala Ala Phe Ala 80 ege aag acc gta gtt gcg ccc 288 Arg 90 Lys Thr Val Val Ala 95 Pro -52- • · · · • · * • * ······· ·· ate gat cac ttt ege ttc aat ggc gcc ggc aag gtg gtg agc ate ege 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 att cac gca tgc cag atg Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gin Met 115 120 125 <210> 22 <211> 125 <212> PRT <213> Sintetični <4 00> 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 Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp Asp 20 25 30 Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr Ala 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 Thr 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 <210> 23 <211> 669 <212> DNK <213> Sintetični <220> <221> CDS <222> (D .. .(669) <223> mNGF <400> 23 gaa ccg tat act gat agc aac gtg cca gaa ggc gat agc gta cca gaa Glu Pro Tyr Thr Asp Ser Asn Val Pro Glu Gly Asp Ser Val Pro Glu 1 5 10 15 gcc cat tgg aca aaa ctg caa cac agc ctg gat aca gca ctg cgt cgt Ala His Trp Thr Lys Leu Gin His Ser Leu Asp Thr Ala Leu Arg Arg 20 25 30 gcc cgt tca gca cca aca get cct att get gcc cgt gtt act ggt cag Ala Arg Ser Ala Pro Thr Ala Pro Ile Ala Ala Arg Val Thr Gly Gin • · -53- 35 40 45 act cgt aat ate acg gtg gat cct cgt ctg ttc aaa aaa ege ege ctg 192 Thr Arg Asn Ile Thr Val Asp Pro Arg Leu Phe Lys Lys Arg Arg Leu 50 55 60 cat tet cct cgt 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 cgt acc cat cgt tca aaa ege tca agc acc cat ccg gtg ttt cat atg 336 Arg Thr His Arg Ser Lys Arg Ser Ser Thr His Pro Val Phe His Met 100 105 110 ggg gaa ttt teg gtt tgt gac agc gtg tet 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 gcg acc gat ate aaa ggc aaa gaa gtg acc gtc ctg gcc gaa 432 Thr Thr Ala Thr Asp Ile Lys Gly Lys Glu Val Thr Val Leu Ala Glu 130 135 140 gtc aat ate aac aac agc gtc ttt cgt caa tat ttc ttc gaa acc aaa 480 Val As n Ile Asn Asn Ser Val Phe Arg Gin Tyr Phe Phe Glu Thr Lys 145 150 155 160 tgc cgt gcc tca aat cct gta gaa agc ggg tgt cgt gga att gat tca 528 Cys Arg Ala Ser Asn Pro Val Glu Ser Gly Cys Arg Gly Ile Asp Ser 165 170 175 aaa cat tgg aac teg tat tgt acc acc act cac acc ttc gtt aaa gcc 576 Lys His Trp Asn Ser Tyr Cys Thr Thr Thr His Thr Phe Val Lys Ala 180 185 190 ctg act acc gac gag aaa caa get get tgg ege ttt ate cgt ate gat 624 Leu Thr Thr Asp Glu Lys Gin Ala Ala Trp Arg Phe Ile Arg Ile Asp 195 200 205 acc get tgt gtg tgt gtc ctg tcc cgt aaa gca aca cgt cgt ggt 669 Thr Ala Cys Val Cys Val Leu Ser Arg Lys Ala Thr Arg Arg Gly 210 215 220 <210> 24 <211> 223 <212> PRT <213> Sintetični <4 00> 24 Glu Pro Tyr Thr Asp Ser Asn Val Pro Glu Gly Asp Ser Val Pro Glu 1 5 10 15 Ala His Trp Thr Lys Leu Gin His Ser Leu Asp Thr Ala Leu Arg Arg 20 25 30 Ala Arg Ser Ala Pro Thr Ala Pro Ile Ala Ala Arg Val Thr Gly Gin 35 40 45Leu 250 Lys Glu Lys Ile Gin 255 Ala Val Gly Val Pro Gly 270 Val Gly Gly Val Pro Gly 285 Val Gly Val Val Pro Gly 300 Val Gly Val Pro Gly Lys 315 Trp Ala Ala Ile Glu 320 Ala 330 Ala Ala Ile Glu Glu 335 Lys Ala Ile Glu Glu Lys 350 Trp Ala Glu Glu Lys Trp 365 Ala Ala Ile Gly Thr Ser 380 gta cag ege ttt gtg get gcg 48 Val 10 Gin Arg Phe Val Ala 15 Ala gtc gcg ctg ttt gcc gat gac 96 Val Ala Leu Phe Ala 30 Asp Asp gag ccc agg tcc ggt acg get 144 Glu Pro Arg Ser 45 Gly Thr Ala ctc aaa ctg cct ttg gcg gtg 192 Leu Lys Leu 60 Pro Leu Ala Val gcc aac gaa gcg gcc ttc get 240 Ala Asn 75 Glu Ala Ala Phe Ala 80 ege aag acc gta gtt gcg ccc 288 Arg 90 Lys Thr Val Val Ala 95 Pro -52- • · · · • · * • * ········· ate gat cac ttt ege ttc aat ggc gcc ggc aag gtg gtg agc ate ege 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 att cac gca tgc cag atg Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gin Met 115 120 125 < 210 > 22 < 211 > 125 < 212 > PRT < 213 > Synthetic < 4 00 > 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 Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp Asp 20 25 30 Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr Ala 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 Thr 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 < 210 > 23 < 211 > 669 < 212 > DNA < 213 > Synthetic < 220 > < 221 > CDS < 222 > (D ... (669) < 223 > mNGF < 400 > 23 gaa ccg tat act gat agc aac gtg cca gaa ggc gat agc gta cca gaa Glu Pro Tyr Thr Asp Ser Asn Val Pro Glu Gly Asp Ser Val Pro Glu 1 5 10 15 gcc cat tgg aca aaa ctg caa cac agc ctg gat aca gca ctg cgt cgt Ala His Trp Thr Lys Leu Gin His Ser Leu Asp Thr Ala Leu Arg Arg 20 25 30 gcc cgt tca gca cca aca get cct att get gcc cgt gtt act ggt cag Ala Arg Ser Ala Pro Thr Ala Pro Ile Ala Ala Arg Val Thr Gly Gin • · -53- 35 40 45 act cgt aat ate acg gtg gat cct cgt ctg ttc aaa aaa ege ege ctg 192 Thr Arg Asn Ile Thr Val Asp Pro Arg Leu Phe Lys Lys Arg Arg Leu 50 55 60 cat tet cct cgt 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 As n 85 90 95 cgt acc cat cgt tca aaa ege tca agc acc cat ccg gtg ttt cat atg 336 Arg Thr His Arg Ser Lys Arg Ser Ser Thr His Pro Val Phe His Met 100 105 110 ggg gaa ttt teg gtt tgt gac agc gtg tet 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 gcg acc gat ate aaa ggc aaa gaa gtg acc gtc ctg gcc gaa 432 Thr Thr Ala Thr Asp Ile Lys Gly Lys Glu Val Thr Val Leu Ala Glu 130 135 140 gtc aat ate aac aac agc gtc ttt cgt caa tat ttc ttc gaa acc aaa 480 Val As n Ile Asn Asn Ser Val Phe Arg Gin Tyr Phe Phe Glu Thr Lys 145 150 155 160 tgc cgt gcc tca aat cct gta gaa agc ggg tgt cgt gga att gat tca 528 Cys Arg Ala Ser Asn Pro Val Glu Ser Gly Cys Arg Gly Ile Asp Ser 165 170 175 aaa cat tgg aac teg tat tgt acc acc act act cac acc ttc gtt aaa gcc 576 Lys His Trp Asn Ser Tyr Cys Thr Thr Thr His Thr Phe Val Lys Ala 180 185 190 ctg act acc gac gag aaa caa get get tgg ege ttt ate cgt ate gat 624 Leu Thr Thr Asp Glu Lys Gin Ala Ala Trp Arg Phe Ile Arg Ile Asp 195 200 205 acc get tgt gtg tgt gtc ctg tcc cgt aaa gca aca cgt cgt ggt 669 Thr Ala Cys Val Cys Val Leu Ser Arg Lys Ala Thr Arg Arg Gly 210 215 220 < 210 > 24 < 211 > 223 < 212 > PRT < 213 > Synthetic < 4 00 > 24 Glu Pro Tyr Thr Asp Ser Asn Val Pro Glu Gly Asp Ser Val Pro Glu 1 5 10 15 Ala His Trp Thr Lys Leu Gin His Ser Leu Asp Thr Ala Leu Arg Arg 20 25 30 Ala Arg Ser Ala Pro Thr Ala Pro Ile Ala Ala Arg Val Thr Gly Gin 35 40 45
Thr Arg Asn Ile Thr Val Asp Pro Arg Leu Phe Lys Lys Arg Arg Leu 50 55 60 His Ser Pro Arg Val Leu Phe Ser Thr Gin Pro Pro Pro Thr Ser Ser 65 70 75 80 Asp Thr Leu Asp Leu Asp Phe Gin Ala His Gly Thr Ile Pro Phe Asn 85 90 95 Arg Thr His Arg Ser Lys Arg Ser Ser Thr His Pro Val Phe His Met 100 105 110 Gly Glu Phe Ser Val Cys Asp Ser Val Ser Val Trp Val Gly Asp Lys 115 120 125 Thr Thr Ala Thr Asp Ile Lys Gly Lys Glu Val Thr Val Leu Ala Glu 130 135 140 Val Asn Ile Asn Asn Ser Val Phe Arg Gin Tyr Phe Phe Glu Thr Lys 145 150 155 160 Cys Arg Ala Ser Asn Pro Val Glu Ser Gly Cys Arg Gly Ile Asp Ser 165 170 175 Lys His Trp Asn Ser Tyr Cys Thr Thr Thr His Thr Phe Val Lys Ala 180 185 190 Leu Thr Thr Asp Glu Lys Gin Ala Ala Trp Arg Phe Ile Arg Ile Asp 195 200 205 Thr Ala Cys Val Cys Val Leu Ser Arg Lys Ala Thr Arg Arg Gly 210 215 220 <210> 25 <211> 186 <212> DNK <213> Sintetični <220> <221> ' CDS <222> (D ·. . (186) <223> . AEA <400> 25 aag tgg get gcc ate gaa gaa gaa gca gcc get att aaa gaa gag gca Lys Trp Ala Ala Ile Glu Glu Glu Ala Ala Ala Ile Lys Glu Glu Ala 1 5 10 15 gca gcg ate gaa gaa aaa gcc gca gcc ate aaa gag gaa gcc gca get Ala Ala Ile Glu Glu Lys Ala Ala Ala Ile Lys Glu Glu Ala Ala Ala 20 25 30 ate gag gaa aaa tgg get gcg att gaa gag gaa get gcg gca ata gaa Ile Glu Glu Lys Trp Ala Ala Ile Glu Glu Glu Ala Ala Ala Ile Glu 35 40 45 gag aaa tgg get get ate aaa gaa aaa gcg get gca ate aaa Glu Lys Trp Ala Ala Ile Lys Glu Lys Ala Ala Ala Ile Lys 50 55 60 48 96 144 186 < 210 > 26 <211> 62 <212> PRT <213> Sintetični <400> 26Thr Arg Asn Ile Thr Val Asp Pro Arg Leu Phe Lys Lys Arg Arg Leu 50 55 60 His Ser Pro Arg Val Leu Phe Ser Thr Gin Pro Pro Pro Thr Ser Ser 65 70 75 80 Asp Thr Leu Asp Leu Asp Phe Gin Ala His Gly Thr Ile Pro Phe Asn 85 90 95 Arg Thr His Arg Ser Lys Arg Ser Ser Thr His Pro Val Phe His Met 100 105 110 Gly Glu Phe Ser Val Cys Asp Ser Val Ser Val Trp Val Gly Asp Lys 115 120 125 Thr Thr Ala Thr Asp Ile Lys Gly Lys Glu Val Thr Val Leu Ala Glu 130 135 140 Val Asn Ile Asn Asn Ser Val Phe Arg Gin Tyr Phe Phe Glu Thr Lys 145 150 155 160 Cys Arg Ala Ser Asn Pro Val Glu Ser Gly Cys Arg Gly Ile Asp Ser 165 170 175 Lys His Trp Asn Ser Tyr Cys Thr Thr Thr His Thr Phe Val Lys Ala 180 185 190 Leu Thr Thr Asp Glu Lys Gin Ala Ala Trp Arg Phe Ile Arg Ile Asp 195 200 205 Thr Ala Cys Val Cys Val Leu Ser Arg Lys Ala Thr Arg Arg Gly 210 215 220 < 210 > 25 < 211 > 186 < 212 > DNA < 213 > Synthetic < 220 > < 221 > 'CDS < 222 > (D ·.. (186) < 223 >. AEA < 400 > 25 aag tgg get gcc ate gaa gaa gaa gca gcc get att aaa gaa gag gca Lys Trp Ala Ala Ile Glu Glu Glu Ala Ala Ala Ile Lys Glu Glu Ala 1 5 10 15 gca gcg ate gaa gaa aaa gcc gca gcc ate aaa gag gaa gcc gca get Ala Ala Ile Glu Glu Lys Ala Ala Ala Ile Lys Glu Glu Ala Ala Ala 20 25 30 ate gag gaa aaa tgg get gcg att gaa gag gaa get gcg gca ata gaa Ile Glu Glu Lys Trp Ala Ala Ile Glu Glu Glu Ala Ala Ala Ala Ile Glu 35 40 45 gag aaa tgg get get ate aaa gaa aaa gcg get gca ate aaa Glu Lys Trp Ala Ala Ile Lys Glu Lys Ala Ala Ala Ile Lys 50 55 60 48 96 144 186 < 210 > 26 < 211 > 62 < 212 > PRT < 213 > Synthetic < 400 > 26
Lys Trp Ala Ala Ile Glu Glu Glu Ala Ala Ala Ile Lys Glu Glu Ala 1 5 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 Ile 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 <210> 27 <211> 99 <212 > DNK <213> Sintetični <220> <221> CDS <222> (1)..(99) <223> Ρ3 <4 00> 27 tcc ccg gaa gat gag ate cag caa ctg gaa gaa gaa ate get cag ctgLys Trp Ala Ala Ile Glu Glu Glu Ala Ala Ala Ile Lys Glu Glu Ala 1 5 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 Ile 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 < 210 > 27 < 211 > 99 < 212 > DNA < 213 > Synthetic < 220 > < 221 > CDS < 222 > (1) .. (99) < 223 > Ρ3 < 4 00 > 27 tcc ccg gaa gat gag ate cag caa ctg gaa gaa gaa ate get cag ctg
Ser Pro Glu Asp Glu Ile Gin Gin Leu Glu Glu Glu Ile Ala Gin Leu 15 10 15 gaa cag aaa aac gca gcg ctg aaa gag aaa aac cag gcg ctg aaa tacSer Pro Glu Asp Glu Ile Gin Gin Leu Glu Glu Glu Ile Ala Gin Leu 15 10 15 gaa cag aaa aac gca gcg ctg aaa gag aaa aac cag gcg ctg aaa tac
Glu Gin Lys Asn Ala Ala Leu Lys Glu Lys Asn Gin Ala Leu Lys Tyr 20 25 30 ggtGlu Gin Lys Asn Ala Ala Leu Lys Glu Lys Asn Gin Ala Leu Lys Tyr 20 25 30 ggt
Gly <210> 28 <211> 33 <212> PRT <213> Sintetični <400> 28Gly < 210 > 28 < 211 > 33 < 212 > PRT < 213 > Synthetic < 400 > 28
Ser Pro Glu Asp Glu Ile Gin Gin Leu Glu Glu Glu Ile Ala Gin Leu 15 10 15Ser Pro Glu Asp Glu Ile Gin Gin Leu Glu Glu Glu Ile Ala Gin Leu 15 10 15
Glu Gin Lys Asn Ala Ala Leu Lys Glu Lys Asn Gin Ala Leu Lys Tyr 20 25 30 48 96 99Glu Gin Lys Asn Ala Ala Leu Lys Glu Lys Asn Gin Ala Leu Lys Tyr 20 25 30 48 96 99
Gly <210> 29 <211> 99 <212> DNK -56- -56-Gly < 210 > 29 < 211 > 99 < 212 > DNA -56- -56-
<213> Sintetični <220> <221> CDS <222> (1)..(99) <223> Ρ4 <400> 29 agc ccg gaa gat aaa att gct cag ctg aaa caa aaa ate caa gcg ctg 48< 213 > Synthetic < 220 > < 221 > CDS < 222 > (1) .. (99) < 223 > Ρ4 < 400 > 29 agc ccg gaa gat aaa att gct cag ctg aaa caa aaa ate caa gcg ctg 48
Ser Pro Glu Asp Lys Ile Ala Gin Leu Lys Gin Lys Ile Gin Ala Leu 15 10 15 aaa cag gaa aac cag cag ctg gaa gag gaa aac gcc gca ctg gaa tat 96Ser Pro Glu Asp Lys Ile Ala Gin Leu Lys Gin Lys Ile Gin Ala Leu 15 10 15 aaa cag gaa aac cag cag ctg gaa gag gaa aac gcc gca ctg gaa tat 96
Lys Gin Glu Asn Gin Gin Leu Glu Glu Glu Asn Ala Ala Leu Glu Tyr 20 25 30 ggt 99Lys Gin Glu Asn Gin Gin Leu Glu Glu Glu Asn Ala Ala Leu Glu Tyr 20 25 30 ggt 99
Gly <210> 30 <211> 33 <212> PRT <213> Sintetični <4 00> 30Gly < 210 > 30 < 211 > 33 < 212 > PRT < 213 > Synthetic < 4 00 > 30
Ser Pro Glu Asp Lys Ile Ala Gin Leu Lys Gin Lys Ile Gin Ala Leu 15 10 15Ser Pro Glu Asp Lys Ile Ala Gin Leu Lys Gin Lys Ile Gin Ala Leu 15 10 15
Lys Gin Glu Asn Gin Gin Leu Glu Glu Glu Asn Ala Ala Leu Glu Tyr 20 25 30Lys Gin Glu Asn Gin Gin Leu Glu Glu Glu Asn Ala Ala Leu Glu Tyr 20 25 30
Gly <210> 31 <211> 99 <212> DNK <213> Sintetični <220> <221> CDS <222> (1)..(99) <223> P5 <400> 31 tet Ser 1 cct Pro gaa Glu gac Asp gaa Glu 5 aac Asn gca Ala gct Ala ctg Leu gaa Glu 10 gag Glu aaa Lys att Ile gca Ala caa Gin 15 ctg Leu 48 aaa Lys caa Gin aag Lys aac Asn 20 gcg Ala gca Ala ctg Leu aaa Lys gaa Glu 25 gaa Glu att Ile caa Gin gca Ala ctg Leu 30 gaa Glu tat Tyr 96 ggc Gly 99 <210> <211> 32 33 -57-Gly < 210 > 31 < 211 > 99 < 212 > DNA < 213 > Synthetic < 220 > < 221 > CDS < 222 > (1) .. (99) < 223 > P5 < 400 > 31 tet Ser 1 cct Pro gaa Glu gac Asp gaa Glu 5 aac Asn gca Ala gct Ala ctg Leu gaa Glu 10 gag Glu aaa Lys att Ile gca Ala caa Gin 15 ctg Leu 48 aaa Lys caa Gin aag Lys aac Asn 20 gcg Ala gca Ala ctg Leu aaa Lys gaa Glu 25 gaa Glu att Ile caa Gin gca Ala ctg Leu 30 gaa Glu tat Tyr 96 ggc Gly 99 < 210 > < 211 > 32 33 -57-
<212> PRT <213 > Sintetični <400> 32< 212 > PRT < 213 > Synthetic < 400 > 32
Ser Pro Glu Asp Glu Asn Ala Ala Leu Glu Glu Lys Ile Ala Gin Leu 15 10 15Ser Pro Glu Asp Glu Asn Ala Ala Leu Glu Glu Lys Ile Ala Gin Leu 15 10 15
Lys Gin Lys Asn Ala Ala Leu Lys Glu Glu Ile Gin Ala Leu Glu Tyr 20 25 30Lys Gin Lys Asn Ala Ala Leu Lys Glu Glu Ile Gin Ala Leu Glu Tyr 20 25 30
Gly <210> 33 <211> 99 <212> DNK <213> Sintetični <220> <221> CDS <222> (1)..(99) <223> P6 <400> 33 agc Ser 1 ccg Pro gaa Glu gat Asp aaa Lys 5 aac Asn gcc Ala gct Ala ctg Leu aaa Lys 10 gag Glu gaa Glu ate Ile cag Gin gcg Ala 15 ctg Leu 48 gaa Glu gaa Glu gaa Glu aac Asn 20 cag Gin gct Ala ctg Leu gaa Glu gag Glu 25 aaa Lys ate Ile gca Ala cag Gin ctg Leu 30 aaa Lys tat Tyr 96 ggt Gly 99 <210> <211> <212> <213> 34 33 PRT Sintetični <400> 34 Ser 1 Pro Glu Asp Lys 5 Asn Ala Ala Leu Lys 10 Glu Glu Ile Gin Ala 15 Leu Glu Glu Glu Asn Gin Ala Leu Glu Glu Lys Ile Ala Gin Leu Lys Tyr 20 25 30Gly < 210 > 33 < 211 > 99 < 212 > DNA < 213 > Synthetic < 220 > < 221 > CDS < 222 > (1) .. (99) < 223 > P6 < 400 > 33 agc Ser 1 ccg Pro gaa Glu gat Asp aaa Lys 5 aac Asn gcc Ala gct Ala ctg Leu aaa Lys 10 gag Glu gaa Glu ate Ile cag Gin gcg Ala 15 ctg Leu 48 gaa Glu gaa Glu gaa Glu aac Asn 20 cag Gin gct Ala ctg Leu gaa Glu gag Glu 25 aaa Lys ate Ile gca Ala cag Gin ctg Leu 30 aaa Lys tat Tyr 96 ggt Gly 99 < 210 > < 211 > < 212 > < 213 > 34 33 PRT Synthetic < 400 > 34 Ser 1 Pro Glu Asp Lys 5 Asn Ala Ala Leu Lys 10 Glu Glu Ile Gin Ala 15 Leu Glu Glu Glu Asn Gin Ala Leu Glu Glu Lys Ile Ala Gin Leu Lys Tyr 20 25 30
Gly <210> 35 <211> 99 <212> DNK <213> Sintetični <220> <221> CDS <222> (1)..(99) -58-Gly < 210 > 35 < 211 > 99 < 212 > DNA < 213 > Synthetic < 220 > < 221 > CDS < 222 > (1) .. (99) -58-
<223> Ρ7 <400> 35 tcc ccg gag gat gag ate cag gcg ctg gaa gaa aag aac gcc cag ctg 48< 223 > Ρ7 < 400 > 35 tcc ccg gag gat gag ate cag gcg ctg gaa gaa aag aac gcc cag ctg 48
Ser Pro Glu Asp Glu Ile Gin Ala Leu Glu Glu Lys Asn Ala Gin Leu 15 10 15 aag cag gaa att gcg gca ctg gaa gag aag aac cag gcc ctg aag tac 96Ser Pro Glu Asp Glu Ile Gin Ala Leu Glu Glu Lys Asn Ala Gin Leu 15 10 15 aag cag gaa att gcg gca ctg gaa gag aag aac cag gcc ctg aag tac 96
Lys Gin Glu Ile Ala Ala Leu Glu Glu Lys Asn Gin Ala Leu Lys Tyr 20 25 30 ggt 99Lys Gin Glu Ile Ala Ala Leu Glu Glu Lys Asn Gin Ala Leu Lys Tyr 20 25 30 ggt 99
Gly <210> 36 <211> 33 <212> PRT <213> Sintetični <400> 36Gly < 210 > 36 < 211 > 33 < 212 > PRT < 213 > Synthetic < 400 > 36
Ser Pro Glu Asp Glu Ile Gin Ala Leu Glu Glu Lys Asn Ala Gin Leu 15 10 15Ser Pro Glu Asp Glu Ile Gin Ala Leu Glu Glu Lys Asn Ala Gin Leu 15 10 15
Lys Gin Glu Ile Ala Ala Leu Glu Glu Lys Asn Gin Ala Leu Lys Tyr 20 25 30Lys Gin Glu Ile Ala Ala Leu Glu Glu Lys Asn Gin Ala Leu Lys Tyr 20 25 30
Gly <210> 37 <211> 99 <212> DNK <213> Sintetični <220> <221> CDS <222> (1)..(99) <223> P8 <400> 37 tcc ccg gaa gac aaa ate get cag ctg aaa gaa gaa aac cag cag ctg 48 Ser Pro Glu Asp Lys Ile Ala Gin Leu Lys Glu Glu Asn Gin Gin Leu 1 5 10 15 gaa caa aag att cag gcc ctg aag gag gaa aac gca get ctg gaa tac 96 Glu Gin Lys Ile Gin Ala Leu Lys Glu Glu Asn Ala Ala Leu Glu Tyr 20 25 30 ggc 99 Gly <210> 38 <211> 33 <212> PRT <213> Sintetični <4 00> 38 -59-Gly < 210 > 37 < 211 > 99 < 212 > DNA < 213 > Synthetic < 220 > < 221 > CDS < 222 > (1) .. (99) < 223 > P8 < 400 > 37 tcc ccg gaa gac aaa ate get cag ctg aaa gaa gaa aac cag cag ctg 48 Ser Pro Glu Asp Lys Ile Ala Gin Leu Lys Glu Glu Asn Gin Gin Leu 1 5 10 15 gaa caa aag att cag gcc ctg aag gag gaa aac gca get ctg gaa tac 96 Glu Gin Lys Ile Gin Ala Leu Lys Glu Glu Asn Ala Ala Leu Glu Tyr 20 25 30 ggc 99 Gly < 210 > 38 < 211 > 33 < 212 > PRT < 213 > Synthetic < 4 00 > 38 -59-
Ser 1 Pro Glu Asp Lys 5 Ile Al a Gin Leu Lys 10 Glu Glu Asn Gin Gin 15 Leu Glu Gin Lys Ile 20 Gin Al a Leu Lys Glu 25 Glu Asn Al a Al a Leu 30 Glu TyrSer 1 Pro Glu Asp Lys 5 Ile Al a Gin Leu Lys 10 Glu Glu Asn Gin Gin 15 Leu Glu Gin Lys Ile 20 Gin Al a Leu Lys Glu 25 Glu Asn Al a Al a Leu 30 Glu Tyr
GlyGly
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