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dc.contributor.advisorMeiring, S. M.
dc.contributor.advisorVan Heerden, E.
dc.contributor.authorVermeulen, Jan-G.
dc.date.accessioned2016-01-06T09:27:12Z
dc.date.available2016-01-06T09:27:12Z
dc.date.copyright2011-11
dc.date.issued2011-11
dc.date.submitted2011-11
dc.identifier.urihttp://hdl.handle.net/11660/1979
dc.description.abstractEnglish: Tissue factor is a transmembrane glycoprotein that functions as the primary initiator of coagulation in response to mechanical or chemical damage. Due to its key position within the coagulation cascade it also plays an important role in the pathology of thrombosis and thrombotic complications associated with cardiovascular disease as well as in non-thrombotic disorders and diseases such as obesity, diabetes, cancer and HIV-AIDS. Recognising the potential in tissue factor inhibition as a novel approach to antithrombotic therapy, our laboratory utilized phage display technology in a previous study, in order to identify a 26 kDa single chain antibody fragment which functionally inhibits human tissue factor. In the current study, the tissue factor inhibitory single chain variable fragment (TFIscFv) was expressed by means of the pIT2 plasmid vector by Escherichia coli HB2151. This expression system was utilised in an up-scale setting in an attempt to improve the TFI-scFv yield. Although functional TFI-scFv was successfully purified from the culture media by means of Protein A affinity chromatography, the process was hampered by large sample volumes, low levels of expression as well as the high cost involved in Protein A purification. Due to an initial focus on improving TFI-scFv yield through the processing of larger sample volumes rather than the improvement of the expression system, immobilised nickel affinity chromatography was investigated as a more cost effective alternative to Protein A affinity chromatography. It was found that the original expression system was incompatible with immobilised nickel affinity chromatography as the protein was expressed into the culture media. The culture media contained nickel chelating elements that stripped the nickel from the column and consequently prevented TFIscFv purification. Subsequently the TFI-scFv gene was isolated, cloned into an over-expression system and modified to redirect the expression to the bacterial cytoplasm. Although TFI-scFv was successfully redirected to the bacterial cytoplasm and purified by means of nickel affinity chromatography, it was found that expression was hampered due to the presence of rare codons. The detrimental effect of rare codons on TFI scFv yield was addressed through the modification of the expression host by the coexpression of the pRARE plasmid as well as by the rare codon optimization of the TFI-scFv gene sequence for expression in E. coli. Although the co-expression of the pRARE plasmid only slightly improved TFI-scFv yield, a sufficient amount of TFIscFv was generated for functional testing. The modified TFI-scFv displayed a similar inhibition effect with reference to the original construct. The rare codon optimisation resulted in a substantial increase in TFI-scFv yield but consequently resulted in the loss of solubility and the production of inclusion bodies. Although the loss of TFIscFv solubility is unwanted, the high level of expression achieved provides an ideal platform for the further development and characterization TFI-scFv in animal thrombosis models.en_ZA
dc.description.abstractAfrikaans: Weefselfaktor is 'n transmembraan glikoproteïen wat dien as die primêre inisieërder van bloedstolling na aanleiding van meganiese- of chemiese skade. As gevolg van die belangrike posisie van weefselfaktor aan die begin van die stollingskaskade, speel dit ook 'n belangrike rol in die patologie van trombose en die trombotiese komplikasies wat geassosier word met kardiovaskulêre siektes, asook siektes soos vetsug, diabetes, kanker en MIV-vigs. As gevolg van die kardinale rol wat weefselfaktor in die bogenoemde siektes speel, besit die inhibisie van weefselfaktor as ‘n benadering tot anti-trombotiese terapie baie potentiaal. In 'n vorige studie het ons navorsingslaboratorium van “phage display” tegnologie gebruik gemaak om ‘n 26 kDa “single chain variable fragment” teen weefselfaktor te identifiseer wat die inisiëringsfunksie van menslike weefselfaktor inhibeer. In die huidige studie was die teenligaam fragment “Tissue factor inhibitory single chain variable fragment - TFI-scFv” geproduseer deur die uitdrukking van die pIT2 vektor deur Escherichia coli HB2151. Hierdie uitdrukkingsisteem is op groter skaal aangewend in ‘n poging om die TFI-scFv opbrengs te verbeter. Alhoewel funksionele TFI-scFv deur middel van Proteïen A affiniteitschromatografie suksesvol vanuit die kultuurmedia gesuiwer was, was die proses bemoeilik deur groot monster volumes, lae vlakke van uitdrukking, sowel as die hoë koste verbonde aan Proteïen A suiwering. As gevolg van 'n aanvanklike fokus op die verbetering van TFI-scFv opbrengs deur middel van die verwerking van groter monster volumes, eerder as om die uitdrukking sisteem te verbeter, was geïmmobiliseerde nikkel affiniteitschromatografie ondersoek as 'n meer koste effektiewe alternatief tot proteïen A affiniteitschromatografie. Daar is gevind dat die oorspronklike uitdrukking sisteem nie versoenbaar met geïmmobiliseer nikkel affiniteitschromatografie was nie aangesien die uitgedrukte proteïen na die kultuurmedia vervoer was. Daar is gevind dat die kultuurmedia nikkel chelerende elemente bevat het wat die nikkel van die kolom afgestroop het. Die teenwoordigheid van nikkel chelerende elemente het gevolglik die suiwering van TFI-scFv deur middel van geïmmobiliseer nikkel affiniteitschromatografie verhoed. Die TFI-scFv geen was geïsoleer en gekloon in 'n hoë opbrengs uitdrukkingsisteem. Die geen was ook gemodifiseer om die lokalisering van die TFI-scFv te skuif van die bakteriële periplasma na die sitoplasma. Hoewel TFI-scFv suksesvol geherlokaliseer was na die bakteriële sitoplasma sowel as suksesvol gesuiwer was deur middel van nikkel affiniteitschromatografie, is daar gevind dat die uitdrukking belemmer was as gevolg van die teenwoordigheid van seldsame kodons in die DNS. Die nadelige effek van die seldsame kodons op TFI-scFv proteïenopbrengs is aangespreek deur die modifisering van die uitdrukkingsgasheer deur middel van die mede-uitdrukking van die pRARE plasmied. Die nadelige effekte van seldsame kodons was ook aangespreek deur middel van die seldsame kodon optimisering van die TFI-scFv DNS vir uitdrukking in E. coli. Hoewel die mede-uitdrukking van die pRARE plasmied die TFI-scFv opbrengs slegs effens verbeter het, was 'n voldoende hoeveelheid TFI-scFv gegenereer vir funksionele analise. Die gemodifiseerde TFIscFv het ‘n soortgelyke inhibisie effek getoon as die oorspronklike konstruk. Die seldsame kodon optimisering het gelei tot 'n aansienlike toename in die TFI-scFv opbrengs, maar gevolglik gelei tot die verlies van oplosbaarheid weens die produksie van proteïen aggregate bekend as “inclusion bodies”. Hoewel die verlies van TFIscFv oplosbaarheid ongewens was, bied die hoë vlakke van uitdrukking wat bereik is 'n ideale platform vir die verdere ontwikkeling en karakterisering van TFI-scFv in trombotiese diermodelle.af
dc.language.isoenen_ZA
dc.publisherUniversity of the Free Stateen_ZA
dc.subjectThromboplastinen_ZA
dc.subjectBlood coagulation factorsen_ZA
dc.subjectBlood -- Coagulationen_ZA
dc.subjectTissue factoren_ZA
dc.subjectThrombosisen_ZA
dc.subjectSingle chain antibody fragmenten_ZA
dc.subjectRare codonsen_ZA
dc.subjectProtein A affinity chromatographyen_ZA
dc.subjectOverexpression systemsen_ZA
dc.subjectCoagulationen_ZA
dc.subjectImmobilised nickel affinity chromatographyen_ZA
dc.subjectInclusion bodiesen_ZA
dc.subjectDissertation (M.Med.Sc. (Haematology and Cell Biology))--University of the Free State, 2011en_ZA
dc.titleCharacterization of a human inhibitory antibody fragment against tissue factoren_ZA
dc.typeDissertationen_ZA
dc.rights.holderUniversity of the Free Stateen_ZA


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