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[{"key": "dc.contributor.author", "value": "Veneskoski, Katja", "language": null, "element": "contributor", "qualifier": "author", "schema": "dc"}, {"key": "dc.date.accessioned", "value": "2010-01-20T09:38:42Z", "language": null, "element": "date", "qualifier": "accessioned", "schema": "dc"}, {"key": "dc.date.available", "value": "2010-01-20T09:38:42Z", "language": null, "element": "date", "qualifier": "available", "schema": "dc"}, {"key": "dc.date.issued", "value": "2009", "language": null, "element": "date", "qualifier": "issued", "schema": "dc"}, {"key": "dc.identifier.other", "value": "oai:jykdok.linneanet.fi:1117987", "language": null, "element": "identifier", "qualifier": "other", "schema": "dc"}, {"key": "dc.identifier.uri", "value": "https://jyx.jyu.fi/handle/123456789/22734", "language": null, "element": "identifier", "qualifier": "uri", "schema": "dc"}, {"key": "dc.description.abstract", "value": "Biotiinia eli vitamiinia H suurimmalla luonnossa tavatulla ei-kovalenttisella affiniteetilla sitovaa avidiinia ja\r\nsen bakteeriper\u00e4ist\u00e4 sukulaisproteiinia streptavidiini k\u00e4ytet\u00e4\u00e4n laajasti erilaisissa avidiini-biotiini teknologian\r\nsovellutuksissa. Alun perin avidiini l\u00f6ydettiin kananmunan valkuaisesta ja sen rakenne on my\u00f6hemmin\r\nselvitetty tarkasti. Avidiinilla ja streptavidiinilla on monia hy\u00f6dyllisi\u00e4 ominaisuuksia. Avidiinin ja\r\nstreptavidiinin on havaittu biotiiniin sitouduttuaan olevan eritt\u00e4in stabiileja proteiineja, jotka kest\u00e4v\u00e4t hyvin\r\nmm. proteolyyttisi\u00e4 entsyymej\u00e4 ja ne omaavat voimakkaan l\u00e4mm\u00f6nkest\u00e4vyyden. Yksi avidiini koostuu\r\nnelj\u00e4st\u00e4 samanlaisesta monomeerist\u00e4, joista jokainen pystyy sitomaan yhden biotiinimolekyylin.\r\nAvidiinin ja streptavidiinin k\u00e4ytt\u00e4misess\u00e4 on kuitenkin rajoituksia johtuen niiden farmakokineetikasta\r\nja immunologisista ominaisuuksista. N\u00e4ihin ongelmiin on pyritty l\u00f6yt\u00e4m\u00e4\u00e4n vastauksia uusista avidiinin\r\nkaltaisista proteiineista, jotka ovat erilaisia mm. fysikaalisilta ja kemiallisilta ominaisuuksiltaan. Avidiniin\r\nkaltaisia proteiineja on l\u00f6ydetty mm. bakteereista, lintujen ja matelijoiden munista, merisiilest\u00e4 sek\u00e4\r\nviimeisimm\u00e4ksi sienest\u00e4. N\u00e4it\u00e4 proteiineja vertailemalla on havaittu proteiinien olevan varsin\r\nkonservatiivisia biotiinia sitovalta aminohapposekvenssins\u00e4 osalta. Proteiinien ominaisuuksia on tutkittu\r\nmy\u00f6s mutaatiolla, jolloin eri aminohapposekvenssej\u00e4 on siirretty proteiinista toiseen. N\u00e4in on pystytty\r\nparantamaan monia avidiinin ominaisuuksia kuten esimerkiksi l\u00e4mm\u00f6nkest\u00e4vyytt\u00e4, s\u00e4ilytt\u00e4m\u00e4ll\u00e4 samalla\r\nbiotiininsitomiskyky.\r\nT\u00e4ss\u00e4 tutkimuksessa tutkittiin Strongylocentrotus purpuratus merisiilen genomista l\u00f6ydetyn geenin\r\nkoodaaman avidiinin kaltaisen proteiinin, strongavidiinin, ominaisuuksia. Strongylocentrotus purpuratus\r\nmerisiilest\u00e4 per\u00e4isin olevasta viidest\u00e4 geenist\u00e4 ainoastaan yht\u00e4 onnistuttiin monistamaan ja sen proteiinia\r\ntuottamaan. Aikaisemmin merisiilest\u00e4 l\u00f6ydettyjen fibropelliinien, jotka my\u00f6s ovat avidiinin kaltaisia\r\nproteiineja, ei ole havaittu sitovan biotiinia. Strongavidiinin kuitenkin havaittiin sitovan voimakkaasti\r\nbiotiinia.\r\nStrongavidiinia onnistuttiin tuottamaan E. coli bakteerissa ja puhdistamaan 2-iminobiotiinikolumnilla.\r\nStrongavidiinin havaittiin muistuttavan avidiinia rakenteeltaan sek\u00e4 monilta ominaisuuksiltaan, kuten\r\nbiotiinin sitomiskyvylt\u00e4\u00e4n. Biotiinin sitomiskyky varmistettiin ITC-mittauksilla (Isothermal titration\r\ncalorimetry). Proteiinin muita ominaisuuksia tutkittiin SDS-PAGEn, ELISAn ja immunoblottauksen sek\u00e4\r\nDot-blottauksen avulla. Strongavidiinin isoelektrisen pisteen voitiin havaita eroavan avidiinin ja\r\nstreptavidiinin isoelektrisest\u00e4 pisteest\u00e4. Eritt\u00e4in matala isoelektrinen piste aiheutti mm. sen, ett\u00e4\r\nimmunoblottausta ei saatu suoritettua strongavidiinille. Strongavidiinin matala isoelektrinen piste (3,91)\r\nn\u00e4ytti aiheuttavan monessa vaiheessa mittausongelmia.\r\nL\u00e4mp\u00f6k\u00e4sitellyiss\u00e4 n\u00e4ytteiss\u00e4 biotiinin sitoutumisen strongavidiinin havaittiin nostavan\r\nstrongavidiinin l\u00e4mm\u00f6nkest\u00e4vyytt\u00e4. Sitoutumattomassa muodossaan l\u00e4mm\u00f6nkest\u00e4vyys n\u00e4ytti heikommalta\r\nkuin avidiinin ja streptavidiinin l\u00e4mm\u00f6nkest\u00e4vyys.\r\nStrongavidiinilla n\u00e4ytt\u00e4\u00e4 esiintyv\u00e4n ristiinreagoimista avidiinin ja streptavidiinin vasta-aineiden kanssa\r\nDot-bolttauksella suoritettuna sek\u00e4 ELISA-mittauksissa. Strongavidiinilla on kolme kysteiinit\u00e4hdett\u00e4 ja kaksi\r\nn\u00e4ist\u00e4 saattaa muodostaa monomeerien sis\u00e4isi\u00e4 rikkisiltoja samalla tavoin kuin avidiinin monomeereiss\u00e4.\r\nSDS-PAGElla ilman \u03b2-merkaptoetanolin pelkist\u00e4v\u00e4\u00e4 vaikututusta suoritetuissa kokeissa, strongavidiini\r\nesiintyi osittain tetrameerisess\u00e4 muodossa. T\u00e4m\u00e4 viittaa siihen, ett\u00e4 strongavidiini muodostaa my\u00f6s\r\nalayksik\u00f6iden v\u00e4lisi\u00e4 rikkisiltoja.", "language": "fi", "element": "description", "qualifier": "abstract", "schema": "dc"}, {"key": "dc.description.abstract", "value": "Avidin and its bacterial analogue streptavidin, which both bind to the vitamin biotin with the highest affinity\r\nfor non-covalent interactions found in nature, are widely used tools in numerous applications in\r\n(strept)avidin-biotin technology. Avidin was first extracted from chicken egg white and its structure is well\r\nknown. Avidin and streptavidin have many useful characteristics. They are very stabile proteins, especially\r\nwhen bound to biotin. Avidin and streptavidin are resistant to proteolytic enzymes and they have high\r\nthermal stability. Avidin contains four identical avidin monomers, which each bind one biotin molecule.\r\nAlthough, avidin and streptavidin have become widely used tools in the life sciences, their\r\npharmacokinetics and immunological defectiveness is limiting this use. These problems have lead to search\r\nof new biotin binding proteins with different physical and chemical properties. Avidin-like proteins have\r\nbeen found for example in bacteria, bird and reptail eggs, sea urchin and recently in mushrooms. Compared\r\nto avidin, avidin-like proteins have very conserved amino acid sequence in their biotin-binding site.\r\nCharacterization of avidin and proteins similar to avidin has been conducted using other avidin-like proteins\r\nas templates in mutations. Modifications have enhanced many properties in avidin, like thermal stability,\r\nwithout affecting biotin binding ability.\r\nIn present study avidin-like protein, named Strongavidin, encoded by a genome sequence from the sea\r\nurchin Strongylocentrotus purpuratus was characterized. Only one gene from original five genes encoding\r\navidin-like proteins from the sea urchin Strongylocentrotus purpuratus produced successfully. Previously\r\ndiscovered avidin-like fibropellins from the sea urchin did not bind to biotin. However, strongavidin was\r\nfound to bind strongly to biotin.\r\nStrongavidin was produced in E. coli bacteria and purified with 2-iminobiotin column. Strongavidin\r\nresembles avidin by its structure and many characteristics, including biotin binding ability. Biotin binding\r\ncapability was verified with Isothermal titration calorimetry (ITC) analysis. Characterization of strongavidin\r\nincluded also SDS-PAGE, ELISA, immunoblotting and Dot-blotting procedures. Strongavidin diverges from\r\navidin and streptavidin by its isoelectric point. Due to low isoelectric point, we were not able to perform\r\nimmunoblotting to strongavidin. In many procedures, low isolectric point (3,91) of strongavidin appeared to\r\ncause problems.\r\nIn heat treatment SDS-PAGE assay strongavidin showed increased thermal stability when bound to\r\nbiotin. Furthermore, in the absence of biotin strongavidin seems to have lower thermal stability than avidin\r\nand streptavidin.\r\nIn cross reactivity analysis conducted with Dot-blotting and ELISA assay with avidin and streptavidin\r\nantibodies, some cross-reactivity was observed with strongavidin. Strongavidin has three cysteine residues\r\nand two of them, according to the known avidin structure, could form an intramonomeric disulfide bridge.\r\nAdditionaly, tetrameric forms were observed in the SDS-PAGE samples boiled in reducing condition in the\r\nabsence of \u03b2-mercaptoetanol, indicating that strongavidin does have intermonomeric bridges.", "language": "en", "element": "description", "qualifier": "abstract", "schema": "dc"}, {"key": "dc.description.provenance", "value": "Submitted by Sinikka Lehto (slehto@cc.jyu.fi) on 2010-01-20T09:38:42Z\nNo. of bitstreams: 1\nURN_NBN_fi_jyu-201001141039.pdf: 1593272 bytes, checksum: dd90b829c3540438aafcfb226c6c2ba7 (MD5)", "language": "en", "element": "description", "qualifier": "provenance", "schema": "dc"}, {"key": "dc.description.provenance", "value": "Made available in DSpace on 2010-01-20T09:38:42Z (GMT). No. of bitstreams: 1\nURN_NBN_fi_jyu-201001141039.pdf: 1593272 bytes, checksum: dd90b829c3540438aafcfb226c6c2ba7 (MD5)\n Previous issue date: 2009", "language": "en", "element": "description", "qualifier": "provenance", "schema": "dc"}, {"key": "dc.format.extent", "value": "86 sivua", "language": null, "element": "format", "qualifier": "extent", "schema": "dc"}, {"key": "dc.format.mimetype", "value": "application/pdf", "language": null, "element": "format", "qualifier": "mimetype", "schema": "dc"}, {"key": "dc.language.iso", "value": "fin", "language": null, "element": "language", "qualifier": "iso", "schema": "dc"}, {"key": "dc.rights", "value": "In Copyright", "language": "en", "element": "rights", "qualifier": null, "schema": "dc"}, {"key": "dc.subject.other", "value": "biotiini", "language": null, "element": "subject", "qualifier": "other", "schema": "dc"}, {"key": "dc.title", "value": "Strongavidiini - biotiinia sitova merisiilest\u00e4 per\u00e4isin oleva avidiinin kaltainen proteiini", "language": null, "element": "title", "qualifier": null, "schema": "dc"}, {"key": "dc.type", "value": "master thesis", "language": null, "element": "type", "qualifier": null, "schema": "dc"}, {"key": "dc.identifier.urn", "value": "URN:NBN:fi:jyu-201001141039", "language": null, "element": "identifier", "qualifier": "urn", "schema": "dc"}, {"key": "dc.type.dcmitype", "value": "Text", "language": "en", "element": "type", "qualifier": "dcmitype", "schema": "dc"}, {"key": "dc.type.ontasot", "value": "Pro gradu -tutkielma", "language": "fi", "element": "type", "qualifier": "ontasot", "schema": "dc"}, {"key": "dc.type.ontasot", "value": "Master\u2019s thesis", "language": "en", "element": "type", "qualifier": "ontasot", "schema": "dc"}, {"key": "dc.contributor.faculty", "value": "Matemaattis-luonnontieteellinen tiedekunta", "language": "fi", "element": "contributor", "qualifier": "faculty", "schema": "dc"}, {"key": "dc.contributor.faculty", "value": "Faculty of Sciences", "language": "en", "element": "contributor", "qualifier": "faculty", "schema": "dc"}, {"key": "dc.contributor.department", "value": "Bio- ja ymp\u00e4rist\u00f6tieteiden laitos", "language": "fi", "element": "contributor", "qualifier": "department", "schema": "dc"}, {"key": "dc.contributor.department", "value": "Department of Biological and Environmental Science", "language": "en", "element": "contributor", "qualifier": "department", "schema": "dc"}, {"key": "dc.contributor.organization", "value": "University of Jyv\u00e4skyl\u00e4", "language": "en", "element": "contributor", "qualifier": "organization", "schema": "dc"}, {"key": "dc.contributor.organization", "value": "Jyv\u00e4skyl\u00e4n yliopisto", "language": "fi", "element": "contributor", "qualifier": "organization", "schema": "dc"}, {"key": "dc.subject.discipline", "value": "Solu- ja molekyylibiologia", "language": "fi", "element": "subject", "qualifier": "discipline", "schema": "dc"}, {"key": "dc.subject.discipline", "value": "Cell and molecular biology", "language": "en", "element": "subject", "qualifier": "discipline", "schema": "dc"}, {"key": "dc.type.coar", "value": "http://purl.org/coar/resource_type/c_bdcc", "language": null, "element": "type", "qualifier": "coar", "schema": "dc"}, {"key": "dc.rights.accesslevel", "value": "openAccess", "language": "fi", "element": "rights", "qualifier": "accesslevel", "schema": "dc"}, {"key": "dc.type.publication", "value": "masterThesis", "language": null, "element": "type", "qualifier": "publication", "schema": "dc"}, {"key": "dc.subject.oppiainekoodi", "value": "4013", "language": null, "element": "subject", "qualifier": "oppiainekoodi", "schema": "dc"}, {"key": "dc.subject.yso", "value": "proteiinit", "language": null, "element": "subject", "qualifier": "yso", "schema": "dc"}, {"key": "dc.subject.yso", "value": "avidiini", "language": null, "element": "subject", "qualifier": "yso", "schema": "dc"}, {"key": "dc.subject.yso", "value": "streptavidiini", "language": null, "element": "subject", "qualifier": "yso", "schema": "dc"}, {"key": "dc.subject.yso", "value": "merisiilit", "language": null, "element": "subject", "qualifier": "yso", "schema": "dc"}, {"key": "dc.format.content", "value": "fulltext", "language": null, "element": "format", "qualifier": "content", "schema": "dc"}, {"key": "dc.rights.url", "value": "https://rightsstatements.org/page/InC/1.0/", "language": null, "element": "rights", "qualifier": "url", "schema": "dc"}, {"key": "dc.type.okm", "value": "G2", "language": null, "element": "type", "qualifier": "okm", "schema": "dc"}]
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