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RECEPTOR MEDIATED ORAL DELIVERY OF BIOENCAPSULATED GREEN FLUORESCENT PROTEIN EXPRESSED IN TRANSGENIC CHLOROPLASTS
Title
RECEPTOR MEDIATED ORAL DELIVERY OF BIOENCAPSULATED GREEN FLUORESCENT PROTEIN EXPRESSED IN TRANSGENIC CHLOROPLASTS.
Creator
Limaye, Arati, Daniell, Henry, University of Central Florida
Abstract / Description
The skyrocketing costs of prescription medicine in developed countries and their lack of availability in developing countries are the most challenging problems of human health. Primary reasons for such high cost are fermentation-based production, expensive purification methods, the need for low temperature storage and transportation and the delivery through sterile injections. Most of these expenses could be minimized or eliminated when therapeutic proteins are expressed and orally delivered...
Show moreThe skyrocketing costs of prescription medicine in developed countries and their lack of availability in developing countries are the most challenging problems of human health. Primary reasons for such high cost are fermentation-based production, expensive purification methods, the need for low temperature storage and transportation and the delivery through sterile injections. Most of these expenses could be minimized or eliminated when therapeutic proteins are expressed and orally delivered via plant cells. Chloroplasts have the machinery to fold complex and biologically active eukaryotic proteins in the soluble chloroplast stromal compartment. Protein expression through chloroplast transformation system offers a number of advantages over nuclear transformation such as a high level of transgene expression (up to 47% of the total soluble protein), due to the presence of 10,000 copies of the transgene per cell, which is uniquely advantageous for oral delivery of adequate amounts of the therapeutic protein or vaccine antigen. It is also an environmentally friendly approach due to effective gene containment and lack of transgene expression in pollen since the chloroplast genome is maternally inherited. To study receptor-mediated oral delivery of therapeutic proteins using the transmucosal carrier cholera toxin B subunit (CTB), a CTB-GFP fusion protein separated by a furin cleavage site was expressed via the tobacco chloroplast genome and used as a visible marker. Site specific integration of the transgene was confirmed by PCR analysis. Southern blot analysis confirmed homoplasmy. Immunoblot analysis confirmed the expression of both the monomeric as well as the pentameric forms of CTB-GFP in transgenic plants. Expression levels of upto 21.3% were obtained and the functionality of the CTB-GFP pentamers was confirmed by an in vitro GM1 binding assay. GFP was seen in the intestinal mucosa, liver and spleen of mice orally fed with CTB-GFP expressing leaves, while CTB was detected only in the intestinal cells. Intestinal macrophages and dendritic cells stained positive for both the CTB as well as GFP. These results suggest successful cleavage of the foreign protein from the transmucosal carrier and its delivery to various organs. These investigations should facilitate the development of a novel cost-effective oral delivery system for plant-derived therapeutic proteins.
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Date Issued
2005
Identifier
CFE0000891, ucf:46633
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0000891
AMYLOID-BETA42 TOXICITY REDUCTION IN HUMAN NEUROBLASTOMA CELLS USING CHOLERA TOXIN B SUBUNIT-MYELIN BASIC PROTEIN EXPRESSED IN CHLOROPLASTS
Title
AMYLOID-BETA42 TOXICITY REDUCTION IN HUMAN NEUROBLASTOMA CELLS USING CHOLERA TOXIN B SUBUNIT-MYELIN BASIC PROTEIN EXPRESSED IN CHLOROPLASTS.
Creator
Ayache, Alexandra, Daniell, Henry, University of Central Florida
Abstract / Description
Alzheimer's disease (AD) is an age progressive neurodegenerative brain disorder, affecting 37 million people worldwide. Cleavage of amyloid precursor protein by β- and γ-secretase produces the amyloid-beta (Aβ) protein, which significantly contributes to AD pathogenesis. The Aβ aggregates, formed at the surface of neurons and intracellularly, cause neurotoxicity and decrease synaptic function. Inhibiting or degrading Aβ accumulation is a key goal for development of new AD treatments. Evidence...
Show moreAlzheimer's disease (AD) is an age progressive neurodegenerative brain disorder, affecting 37 million people worldwide. Cleavage of amyloid precursor protein by β- and γ-secretase produces the amyloid-beta (Aβ) protein, which significantly contributes to AD pathogenesis. The Aβ aggregates, formed at the surface of neurons and intracellularly, cause neurotoxicity and decrease synaptic function. Inhibiting or degrading Aβ accumulation is a key goal for development of new AD treatments. Evidence shows that human Myelin Basic Protein (MBP) binds to and degrades Aβ thereby, preventing cytotoxicity. A potential method for oral drug delivery that will allow plant-derived bioencapsulated MBP to pass through intestinal epithelium and bypass denaturing stomach acidity is quite novel. Cholera Toxin B subunit (CTB), when fused with MBP, can serve as a vehicle for oral delivery of this chloroplast expressed therapeutic protein into the systemic circulation. Within chloroplast, CTB forms a pentameric structure that binds to GM1 ganglioside receptors, allowing receptor-mediated endocytosis. In order to investigate protein entry through neuronal GM1 receptors, we first created CTB fused to the green fluorescent protein (GFP). Incubation of this fusion protein with human neuroblastoma cells resulted in GFP entry into these cells whereas GFP alone was unable to enter. Similarly, co-incubation of CTB-MBP, via neuronal GM1 binding, allowed MBP to reduce neurotoxicity of Aβ42 treated cells by 37.1%. Delivery of CTB-MBP through GM1 receptor mediated binding should therefore facilitate oral administration, storage, heat stability and low cost AD treatment.
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Date Issued
2012
Identifier
CFH0004249, ucf:44916
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFH0004249