Chemical and Biomolecular Engineering Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2751
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Item Expression and Purification of the Cell-Penetrating Peptide MAP Fused to Protein Cargo(2024) CHUNG, REN-JHE; Karlsson, Amy J; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Candida albicans is a common fungal pathogen that presents a significant public health issue due to its resistance to traditional antifungal small molecule medications. While large therapeutics offer potential as alternative treatments with novel mechanisms of action, their efficacy is hindered by low cellular uptake and limited accessibility to intracellular targets. Cell-penetrating peptides (CPPs) are promising vehicles for facilitating the intracellular delivery of large biomolecules. CPPs are short protein peptides capable of crossing various membrane barriers, even when conjugated to cargo molecules. However, producing CPP fusion proteins can be challenging due to their higher toxicity to host cells. To enhance expression, we studied and optimized several contributing factors, including expression partners, host cell strains, sequential order of CPP and cargo, and induction conditions, for the fusion of CPP MAP to the cargo monomeric enhanced green fluorescent protein (meGFP). Our findings indicated that the expression partner, combined with positioning MAP at the N-terminus of the cargo, resulted in relatively high expression levels. The highest expression level was achieved in the BL21(DE3) Escherichia coli strain, with induction at 20°C for 24 hours. These results support further research on the application of MAP recombinant proteins and lay the foundation for the generalization of CPP recombinant protein expression.Item Sutureless Anastomosis: Electroadhering a Hydrogel Sleeve Over Cut Pieces of Tubular Tissue(2024) Grasso, Samantha Marie; Raghavan, Srinivasa R; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Recently, our lab demonstrated that cationic gels could be adhered to animal tissues by applying an electric field (10 V DC, for ~ 20 s). This phenomenon, termed electroadhesion (EA), could potentially be used to repair injured tissues without sutures. An extreme injury is when a tube in the body (e.g., a blood vessel or an intestine) is cut into two segments. The surgical process of joining the segments is termed anastomosis, and thus far has only been done clinically with sutures. Here, we explore the use of EA for performing sutureless anastomoses in vitro with bovine aorta and chicken intestine. For this purpose, we make a strong and stretchable cationic gel in the form of a sleeve (i.e., a hollow tube). By using a custom plastic mold, we control both the sleeve diameter and wall thickness. A sleeve with a diameter matching that of the tubular tissue is slipped over the cut segments of the tube, followed by application of the DC electric field. Thereby, the sleeve becomes strongly adhered by EA to the underlying tube. Water or blood is then flowed through the repaired tube, and we record the burst pressure Pburst of the tube. We find that Pburst is > 80 mm Hg and close to the Pburst of an intact (uncut) tube. In comparison to the sleeve, a long strip of the gel attached around the cut tubular pieces allows a much lower Pburst. Thus, our study shows that gel-sleeves adhered by EA could enable anastomoses to be performed in the clinic without the need for sutures.Item EXPERIMENTAL INVESTIGATION OF THE LIPID-BINDING MECHANISM OF OSH4 PROTEIN(2024) Konakbayeva, Dinara; Karlsson, Amy; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Recent findings show that intracellular lipid traffic between organelles primarily occurs through a non-vesicular pathway involving lipid transport proteins (LTPs) and is facilitated by areas of close apposition between two organelles so called membrane contact sites (MCS). Oxysterol-binding homologue (Osh) proteins in the yeast Saccharomyces cerevisiae serve as examples of LTPs. Osh proteins are crucial for transporting signaling lipids and are believed to form MCSs. In this study, we examined the binding mechanism of the Osh4 protein, aiming to gain a better understanding of its explicit membrane-binding mechanism.The Osh4 protein possesses an α-helical binding domain known as the amphipathic lipid-packing sensor (ALPS)-like motif. Our approach involved utilizing experimental methods to examine the biophysical interactions of both the ALPS peptide and the full-length Osh4 protein. To investigate the binding interactions of ALPS with membranes of different lipid compositions, we examined its interactions with three different mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC; has a zwitterionic head group) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS; has a negatively charged head group)—1:1 POPC-POPS, 4:1 POPC-POPS, and 9:1 POPC-POPS—as well as pure POPC. To understand the structural changes in ALPS and model membranes during peptide-membrane interactions, we performed a series of experimental studies. Circular dichroism (CD) was used to study the changes in the secondary structure of ALPS in different environments. The CD data indicated that the α-helical conformation of the ALPS peptide was more pronounced in the presence of POPC-POPS liposomes, especially with a higher content of POPS lipid, compared to liposomes composed entirely of POPC. This observation underscores the significant influence of anionic lipids in the facilitation of peptide folding at the membrane-water interface. X-ray diffraction was utilized to study the changes in membrane structure upon ALPS binds to it. The X-ray diffraction results showed that the ALPS peptide caused thinning of the multilayer with an increased POPS lipid ratio. This could be due to the electrostatic interaction of the positively charged Lys residue in the ALPS sequence with the anionic POPS lipid. We also studied the binding of the peptide to membranes by observing changes in the Trp fluorescence emission spectrum of ALPS upon the addition of liposomes. We observed a blue shift in the fluorescence emission maximum of Trp with higher POPS content. This suggests that the ALPS peptide was experiencing a more hydrophobic and less polar environment in the presence of the liposomes, indicating possible penetration of the peptide into the hydrocarbon region of the bilayer. The blue shifts of Trp emission in the presence of POPS liposomes were higher than those observed with POPC liposomes and suggest that the ALPS peptide binds better to charged POPS lipids, which is consistent with the X-ray diffraction data. We also conducted Trp fluorescence titration and ITC experiments to gain deeper insights into the binding affinity of the ALPS peptide to a model membrane. Using fluorescence data, we estimated the binding constant for the binding of ALPS to liposomes by performing titration measurements of vesicles with the ALPS peptide. Our analysis demonstrated that ALPS binding to 4:1 POPC-POPS lipid membranes had a Kd of 1.88 ± 0.47 μM, which corresponds to a free energy change (ΔG) of -7.82 ± 0.15 kcal/mol. Additionally, the ITC experiments performed with the same vesicles yielded a ΔG of -4.41± 0.04 kcal/mol. This result is slightly less than the ΔG value of -7.82 ± 0.15 kcal/mol obtained from fluorescence spectroscopy titration. The observed discrepancy of -3.41 kcal/mol may indicate the energy associated with the folding of the ALPS peptide. In order to understand how Osh4 forms MCSs between two membranes, we need to examine how the membranes interact with the full-length protein. The first step to achieve this is to produce the protein through recombinant protein production methods. After evaluating two different fusion tags, glutathione S-transferase (GST) and small ubiquitin-related modifier (SUMO), it was found that the SUMO tag resulted in higher protein yield and greater protein purity. Our work lays the foundation for future experiments with the full-length Osh4 protein to improve our understanding of the mechanisms of lipid transport between membranes. Our results emphasize the ALPS peptide’s selectivity for specific lipid environments, particularly its affinity for anionic lipids. We demonstrated that the presence of anionic lipids is crucial for the motif's ability to induce conformational changes upon binding to a membrane, and these conformational changes likely play a critical role in intracellular lipid trafficking and membrane organization.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 AN INTEGRATIVE EXPERIMENTAL AND COMPUTATIONAL FRAMEWORK FOR THE GENOME-SCALE FLUX ANALYSIS OF ANTIBIOTIC RESISTANCE(2020) Mack, Sean; Dwyer, Daniel J; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The prevalence of antibiotic-resistant bacterial pathogens demands the development of novel therapeutic approaches. An attractive target is metabolism, where the impact of antibiotics and resistance remains poorly understood. Numerous omics-driven studies have identified a metabolic response due to antibiotic stress and suggested that metabolism adjusts to accommodate the genetic burden of resistance. Arising from these data is the hypothesis that context-specific modification of metabolism is a key component of antibiotic resistance and stress. Further exploration of the relationship between metabolism, antibiotic stress, and resistance is clearly needed. To elucidate metabolic signatures of antibiotic resistance, we analyzed the metabolic behaviors of wild-type and resistant strains of Escherichia coli through a combined transcriptomic and fluxomic analysis. Specifically, we compared wild-type E. coli to isogenic strains expressing integrated copies of tetRA and dhfr resistance genes, respectively under normal and antibiotic stress conditions. From comprehensive genome-scale (GS) flux predictions, we observed a resistance-associated metabolic phenotype as well as mechanism- and target-specific metabolic shifts. Furthermore, we identified a distinct metabolic response to antibiotic stress in both resistant strains. To improve our computational framework, we developed NetRed, NetRed-MFA, and NetFlow, each designed to reduce complexity of GS flux analysis. Through lossless reduction of genome-scale models (GSMs), NetRed generated a comprehensive minimal model for aerobic and anaerobic growth in E. coli and rapidly elucidated the mechanism driving artemisinin production in yeast. NetRed-MFA extended the original algorithm by incorporating full carbon mapping to generate reduced models for 13C metabolic flux analysis. NetFlow leveraged GS carbon mapping to isolate the major carbon flows through a core network and GSM; from the GSM subnetwork, we identified a mechanistic relationship between a triple-knockout and increased lycopene production in E. coli. Our resistance work represents the first application of quantitative flux analysis to study the metabolism of resistant bacteria and should provide significant insight into the role of metabolic adaptation in antibiotic resistance. The developed tools each dramatically improve the interpretation of GS flux predictions and the mechanistic understanding of metabolic perturbations. Taken together, this dissertation describes a comprehensive framework for the prediction, comparison, and interpretation of altered metabolic states.