EVALUATING MODIFIED POLYHEDRIN PROMOTERS FOR IMPROVED CO-EXPRESSION OF KRAS4b
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Type of Work83 Pages
DepartmentHood College Biology
ProgramHood College Biomedical and Environmental Science
Baculovirus Expression Vector System (BEVS)
While 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.