EVALUATING MODIFIED POLYHEDRIN PROMOTERS FOR IMPROVED CO-EXPRESSION OF KRAS4b

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dc.contributor.advisorEsposito, Dominic
dc.contributor.advisorSmith, Oney
dc.contributor.advisorBoyd, Ann
dc.contributor.authorPutman, Zoe
dc.contributor.departmentHood College Biologyen_US
dc.contributor.programHood College Biomedical and Environmental Scienceen_US
dc.date.accessioned2020-11-15T21:05:49Z
dc.date.available2020-11-15T21:05:49Z
dc.date.issued2020-11-15
dc.description.abstractWhile the baculovirus expression vector system (BEVS) has proved its ability to produce high quality recombinant proteins utilizing a eukaryotic host, limited improvements have been made with regards to expressing multiple proteins. Current co-expression protocols utilize native baculovirus promoters which have limited versatility and varying expression levels. This thesis work aims to address these limitations, through the evaluation of modified AcMNPV polyhedrin promoters with the end goal of identifying novel promoters that display varying levels of expression and provide greater control over the ratio of recombinant proteins produced in the cell. Initial promoter screening measured fluorescence output of an eGFP reporter. Promoters of the most interest were then evaluated for test protein expression to determine their ability to produce more complex proteins. Mutations within the minimal promoter region which disrupt critical promoter elements were found to severely impair promoter activity. On the other hand, those which altered the upstream region beyond the transcription start site had little influence on reporter gene levels. Mutations to the polyhedrin promoter CAGT motif which unexpectedly introduced an alternative translation start site (CAtg), appeared to significantly impact downstream gene expression. When this mutation was in-frame with the reporter gene, eGFP expression levels were found to exceed that of the native promoter. Conversely, minimal eGFP expression was observed when this mutation was not in-frame, suggesting potential use of uORFs to regulate expression. Findings from this work will aid future improvements to the BEVS and will be particularly useful for those seeking to improve co-expression protocols.en_US
dc.format.extent83 Pagesen_US
dc.genreThesisen_US
dc.identifierdoi:10.13016/m2theb-1xwt
dc.identifier.urihttp://hdl.handle.net/11603/20062
dc.language.isoen_USen_US
dc.relation.isAvailableAtHood College
dc.subjectProtein expressionen_US
dc.subjectBaculovirusen_US
dc.subjectPolyhedrin promoteren_US
dc.subjectBaculovirus Expression Vector System (BEVS)en_US
dc.subjectInsect Expressionen_US
dc.subjectKRASen_US
dc.titleEVALUATING MODIFIED POLYHEDRIN PROMOTERS FOR IMPROVED CO-EXPRESSION OF KRAS4ben_US
dc.typeTexten_US

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