Structure-Function Studies of Recombinant Apolipoproteins
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Authors
Lethcoe, Kyle
Issue Date
2024
Type
Dissertation
Language
Keywords
Apolipoprotein , Bioreactor , Foam Fractionation , Nanodisk , Nanoparticles , Recombinant Expression
Alternative Title
Abstract
Bioreactor-based foam fractionation is a method developed for the expression and purification of apolipoprotein alpha helix bundle domains for their use as nanoparticle scaffold protein components. Nanodisks (ND) are phospholipid nanoparticles comprised of a bilayer forming phospholipid and an amphipathic apolipoprotein scaffold, which circumscribes the exposed fatty acyl tails of the bilayer, conferring aqueous solubility to the complex. NDs comprised of foam fractionated apolipoproteins were used in a variety of studies to better understand the role of the amphipathic alpha helix bundle domain on cellular escape, foam fractionation potential, and ND stability. Recombinant apolipoproteins comprised entirely of an amphipathic alpha helix bundle such as apolipophorin III (apoLp-III) have previously been shown to escape their bacterial cell hosts when targeted to the periplasmic space using an N-terminal pelB leader signal sequence directly fused to the protein coding sequence. This characteristic was evaluated using other members of the exchangeable apolipoprotein family who contain an N-terminal alpha helix bundle domain in their tertiary protein structure. It was found that introducing C-terminal truncations into the protein coding sequence for recombinant apolipoprotein A-I (apoA-I (1-184)) and apolipoprotein E (apoE4-NT) resulted in cellular escape when directed to the periplasmic space. When conducting recombinant expression of these secreted apolipoproteins in a bioreactor setting, it was observed that formation of a thick foam appeared only after induction with isopropyl β-D-1-thiogalactopyranoside (IPTG). This phenomenon is specific to secreted apolipoprotein amphipathic alpha helix bundle proteins and is directly dependent upon a high concentration of secreted protein. Upon further investigation, I was concluded that foam produced during bioreactor-based expression contained the desired apolipoprotein as its sole major component. It was determined this foam could be fractionated into a separate collection vessel, and upon analysis, the protein produced in this manner contained no negative modifications as a result of the process. Ultimately, the novel foam fractionation purification method takes advantage of the natural biosurfactant like properties of alpha helix bundle apolipoproteins. The helix bundle is amphipathic in nature meaning the protein will preferentially bind free lipid in solution to stabilize its structure. It is believed that when the apolipoprotein is expressed in a bioreactor, the lipid-free protein found in the culture medium will bind microbubbles produced within the bioreactor chamber resulting in formation of a protein stabilized foam. This interaction is facilitated by the hydrophobic amino acid residues of the bundle core interacting with the gas component of the microbubble, and conversely, the hydrophilic amino acid residues interact with residual media forming a liquid interface between individual bubbles. Once a sufficient number of apolipoproteins have bound to the bubble surface, the stabilizing force produced is enough to prevent the bubble from bursting once it reaches the air/liquid interface of the culture medium and foam is produced. Foam fractionated protein was then used as a ND scaffold protein to investigate the lipid binding properties of the isolated protein. ND were formulated using a synthetic phospholipid, dimyristoylphosphatidylcholine (DMPC), and foam fractionated apolipoproteins (apoA-I (1-184), apoE4-NT, and apoLp-III). All three foam fractionated apolipoproteins were found to preferentially bind free DMPC in solution resulting in formation of stable NDs. Successful binding of an apolipoprotein scaffold to suitable lipid results in a dramatic clarification of the ND sample. This event also results in a molecular weight shift that can be visualized using size exclusion chromatography. Each of the apolipoproteins used for ND characterization studies had a characteristic elution peak of ~100-200 kDa, a drastic increase to the lipid-free apolipoprotein molecular weight and is indicative of successful ND formation. Further characterization of NDs constructed using foam fractionated scaffold protein was conducted using the cholesterol efflux capacity assay. This assay is a measure of total cholesterol efflux as a function of present lipid-free and lipid-bound apolipoproteins. It was found that lipid-free apoA-I (1-243; control), apoA-I (1-184), and apoE4-NT showed minimal cholesterol efflux when presented to differentiated THP-1 macrophage cells. Conversely, when incubating cells with NDs comprised of the same apolipoproteins, not only was the efflux value statistically higher than that of their lipid-free counterparts, but both C-terminal truncated variations (apoA-I (1-184) and apoE4-NT) had no statistical difference between particles comprised of a wild type apoA-I (1-243; control) protein. This is further evidence that the amphipathic alpha helix bundle is necessary for ND formation and NDs produced using these proteins exhibited definitive cholesterol efflux capacity. Characterized NDs were further evaluated as drug delivery vehicles for small hydrophobic molecules using the macrolide polyene antibacterial molecule, amphotericin-b (ampB). AmpB is a popular antifungal drug used to treat a variety of fungal infections in humans. The drawback of ampB treatment is the molecule becomes toxic at high concentrations over time and with the emergence of ampB resistant fungi on the rise, a new treatment regimen is required. NDs comprised of DMPC and foam fractionated apoE4-NT were investigated as a potential delivery vehicle of ampB. It was found that NDs enriched with ampB were not only able to drastically increase the solubility of the hydrophobic molecule, but also retained the molecules capacity to inhibit fungal growth. Fungal growth was measured using a yeast viability assay where the ampB NDs produced exhibited a strong growth inhibition with concentrations as low as 10 ng. The retention of biological activity suggests that foam fractionated apolipoprotein NDs represent a viable alternative to current drug delivery formulations. These data presented in this dissertation outline the practical use of foam fractionation for the production and purification of helix bundle apolipoproteins and present a possible alternative to conventional ND formulations for their utility as therapeutic nanoparticles.