Engineering yeast strains for the expression of South African G9P[6] rotavirus VP2 and VP6 structural proteins

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dc.contributor.advisorO’Neill, Hester G.
dc.contributor.advisorAlbertyn, Jacobus
dc.contributor.authorMakatsa, Mohau Steven
dc.date.accessioned2015-11-16T09:44:09Z
dc.date.available2015-11-16T09:44:09Z
dc.date.issued2015-02
dc.description.abstractEnglish: In this study, sequences encoding VP2 and VP6 rotavirus structural proteins from rotavirus strain RVA/Human-wt/ZAF/GR10924/1999/G9P[6] were used in construction of wide-range yeast expression vectors containing the ORFs encoding rotavirus structural proteins VP2 and VP6. VP2 and VP6 sequences were codon-optimized for expression in yeast strains K. lactis, A. adeninivorans and Pichia pastoris/Hansenula polymorpha. Wide-range yeast expression vectors containing either VP6 or VP2 yeast optimized ORFs were constructed for expression of single proteins in different yeast strains. Dual vectors containing both VP6 and VP2 yeast optimized ORFs were constructed to allow simultaneous expression of proteins in different yeast strains and to enable the formation of double-layered rotavirus-like particles. A total of eight yeast strains namely: Kluyveromyces marxianus, Kluyveromyces lactis, Debaryomyces hansenii, Yarrowia lipolytica, Hansenula polymorpha, Pichia pastoris Candida deformans and Arxula adeninivorans were selected for screening. Saccharomyces cerevisiae was included as a positive control as triple-layered rotavirus virus-like particles (tlRLPs) have been successfully produced in this yeast before. All nine yeast strains were successfully transformed with pKM177 vectors containing yeast codon-optimized VP6 ORFs. However, only six yeast strains indicated positive VP6 ORF integration. The other three yeast strains indicated no VP6 integration but hygromycin B integration suggesting that the vectors successfully integrated in these yeast strains but VP6 ORF was lost. Integration of both rotavirus VP2/6 ORFs was evident in all yeast strains transformed with the dual expression vectors. A control to monitor rotavirus VP6 protein expression in yeast was successfully prepared in bacterial cells. The positive control indicated specific reaction when polyclonal rotavirus antibody raised against Nebraska calf diarrhoea rotavirus strain. Rotavirus VP6 expression in yeast strains shown no reactions for K. lactis and A. adeninivorans codon-optimized VP6 ORF in all six yeast strains that tested positive for intergration of the VP6 ORF. All yeast strains except K. marxianus showed possible reaction for VP6 expression for P. pastoris/H. polymorpha codon-optimized VP6 ORF although the identity of VP6 should be confirmed.en_ZA
dc.description.abstractAfrikaans: In hierdie studie, basispaaropeenvolgings wat kodeer vir VP2 en VP6 rotavirus strukturele proteïene van rotavirus stam RVA/Human-wt/ZAF/GR10924/1999/G9P [6] is gebruik in die konstruksie van ‘n wye verskeidenheid gis uitdrukkingsvektore met rotavirus strukturele proteïene VP2 en VP6 oopleesrame. VP2 en VP6 basispaaropeenvolgings is kodon geoptimaliseer vir uitdrukking in gis stamme K. lactis, A. adeninivorans en P. pastoris/H. polymorpha. Wye verskeidenheid gis uitdrukkingsvektore wat geoptimaliseerde oopleesrame van VP6 of VP2 bevat, is gebou vir uitdrukking van enkel proteïene in verskillende gisstamme. Vektore wat beide VP6 en VP2 geoptimaliseerde oopleesrame bevat is gebou vir gelyktydige uitdrukking van proteïene in verskillende gisstamme en om die vorming van dubbellaag rotavirus-agtige partikels toe te laat. 'n Totaal van agt gisstamme naamlik: Kluyveromyces marxianus, Kluyveromyces lactis, Debaryomyces hansenii, Yarrowia lipolytica, Hansenula polymorpha, Pichia pastoris, Candida deformans en Arxula adeninivorans is gekies vir sifting. Saccharomyces cerevisiae is ingesluit as 'n positiewe kontrole aangesien trippellaag rotavirus-agtige partikels (tlRLPs) voorheen suksesvol in hierdie gis geproduseer is. Al nege gisstamme is suksesvol getransformeer met pKM177 vektore wat gis geoptimaliseerde kodon VP6 oopleesrame bevat. Daar was egter slegs ses gisstamme waarin positiewe VP6 ORF integrasie gevind is. Die oorblywende drie gisstamme het higromisien B integrasie getoon, wat daarop dui dat die vektore suksesvol geïntegreer het in hierdie gisstamme, maar dat VP6 oopleesraam verloor is. Integrasie van beide rotavirus VP2/6 oopleesrame was suksesvol vir al die gis stamme wat met die dubbele uitdrukkingsvektore getransformeer is. Rotavirus VP6 proteïenuitdrukking is suksesvol uitgevoer in bakteriële selle. Die positiewe kontrole het ʼn spesifieke reaksie getoon met poliklonale rotavirus teenliggaampies wat teen Nebraska calf diarrhoea rotavirus opgewek is, maar geen reaksie is waargeneem wanneer groep-spesifieke muis monoklonale teenliggaampies gebruik is nie. Rotavirus VP6 uitdrukking in gisstamme het geen reaksies getoon vir K. lactis en A. adeninivorans geoptimiseerde VP6 oopleesrame nie in al ses gisstamme wat positief getoets het vir integrasie van die VP6 oopleesrame. Alle gis stamme behalwe K. marxianus het moontlike reaksie vir VP6 uitdrukking vir P.pastoris / H. polymorpha geoptimiseerde VP6 oopleesraam, alhoewel die identiteit van VP6 nog bevestig moet word.
dc.description.sponsorshipNational Research Foundation (NRF)en_ZA
dc.description.sponsorshipPoliomyelitis Research Foundation (PRF)en_ZA
dc.identifier.urihttp://hdl.handle.net/11660/1665
dc.language.isoenen_ZA
dc.publisherUniversity of the Free Stateen_ZA
dc.rights.holderUniversity of the Free Stateen_ZA
dc.subjectRotavirusen_ZA
dc.subjectVaccineen_ZA
dc.subjectDouble-layer particlesen_ZA
dc.subjectWestern blot analysisen_US
dc.subjectYeasten_ZA
dc.subjectExpressionen_ZA
dc.subjectVectoren_ZA
dc.subjectRotavirus VP2en_ZA
dc.subjectRotavirus VP6en_ZA
dc.subjectOpen reading frameen_ZA
dc.subjectDissertation (M.Sc. (Microbial, Biochemical and Food Biotechnology))--University of the Free State, 2015en_ZA
dc.subjectReovirusesen_ZA
dc.titleEngineering yeast strains for the expression of South African G9P[6] rotavirus VP2 and VP6 structural proteinsen_ZA
dc.typeDissertationen_ZA
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