Chemical and Biomolecular Engineering Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2751
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Item Translocation of protein cargo into Candida albicans using cell-penetrating peptides(2020) Adhikari, Sayanee; Karlsson, Amy J; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Fungal infections caused by Candida albicans pose a serious threat to public health. The rising drug resistance towards azoles, the current first-line antifungal treatment, warrants novel approaches to designing and delivering new antifungal agents that target C. albicans cells. To increase the intracellular delivery of bioactive molecular cargo, we studied the use of cell-penetrating peptides (CPPs) as delivery vehicles. CPPs have been extensively used to deliver different cargoes into mammalian cells, but limited work has focused on delivery into fungal cells. To improve understanding of CPP-mediated delivery to C. albicans, we studied their ability to deliver green fluorescent protein (GFP) intracellularly. For our work, we chose the CPPs, MPG and Hst5, that have previously shown translocation into fungal cells without cargo and recombinantly produced these CPP fusions to GFP in Escherichia coli. We investigated the CPP-mediated translocation of GFP using flow cytometry. Fusion of GFP to MPG resulted in translocation into 40% of C. albicans cells, which was significantly higher than 13% cells that demonstrate translocation of GFP without a CPP. However, Hst5 did not translocate GFP into cells, with only 5% of cells exhibiting Hst5-GFP translocation. Our results demonstrate that MPG can deliver GFP, while Hst5 is not as promising. These results are consistent with molecular dynamics simulations that show MPG enters a model membrane preferentially with the N-terminal residues, whereas Hst5 fails to enter the membrane. Our results emphasize the potential of CPPs in delivering large cargo to C. albicans cells and the advantage of using both experiments and simulations to study the translocation of CPPs into C. albicans. To explore factors affecting translocation efficacy, we evaluated the aggregation of CPP-GFP fusion constructs. Using dynamic light scattering and interference scattering microscopy, our results identified aggregation of our fusions at high concentration as a possible limitation to translocation, motivating future studies of the causes of aggregation and its relationship to translocation efficiency. Our work has shown that CPPs can deliver large biomolecular cargo into fungal cells and has laid the foundation for further studies to design better CPPs and to explore mechanisms limiting translocation of CPPs into fungal pathogens.Item OPTIMIZATION OF RECOMBINANT PROTEIN EXPRESSION FOR CELL-PENETRATING PEPTIDE FUSIONS TO PROTEIN CARGO(2017) Adhikari, Sayanee; Karlsson, Amy J; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Recombinant production of cell-penetrating peptides (CPPs) as fusions to protein “cargo” leads to low yields for some CPP-cargo fusions; thus, ways to enhance the recombinant expression of peptide-cargo fusions need to be identified. We optimized expression conditions for fusions of five CPPs (NPFSD, pVEC, SynB, histatin-5 and MPG) to the cargo proteins biotin carboxyl carrier protein (BCCP), maltose binding protein (MBP) and green fluorescent protein GFP. Glutathione-S-transferase was incorporated as a fusion partner to improve expression. In general, expression at 37 oC for 6 h and 10 h led 2 to the highest levels of expression for the different CPP-cargo constructs. The fusion of histatin-5 to GFP was purified, and its translocation into the fungal pathogen Candida albicans was studied. The purified protein translocated into the nearly 3% of C. albicans cells. These results provide the foundation for future studies to improve translocation of varied CPP-cargo fusions into C. albicans cells.