Current Search: Cholera Toxin B Subunit (x)
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(1 - 3 of 3)
- Title
- EXPRESSION OF HETEROLOGOUS PROTEINS IN TRANSGENIC TOBACCO CHLOROPLASTS TO PRODUCE A BIOPHARMACEUTICAL AND BIOPOLYMER.
- Creator
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Devine, Andrew, Daniell, Henry, University of Central Florida
- Abstract / Description
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The chloroplast has been demonstrated to be an ideal compartment to accumulate certain proteins or their biosynthetic products that would be harmful if they were accumulated in the cytoplasm. Hyper-expression of foreign proteins in chloroplast transgenics has accumulated up to 46% total soluble protein, this is possible due to the ~100 chloroplast genomes per chloroplast and ~100 chloroplasts per cell which can therefore, contain up to 10,000 copies of the transgene. Maternal gene inheritance...
Show moreThe chloroplast has been demonstrated to be an ideal compartment to accumulate certain proteins or their biosynthetic products that would be harmful if they were accumulated in the cytoplasm. Hyper-expression of foreign proteins in chloroplast transgenics has accumulated up to 46% total soluble protein, this is possible due to the ~100 chloroplast genomes per chloroplast and ~100 chloroplasts per cell which can therefore, contain up to 10,000 copies of the transgene. Maternal gene inheritance of plastids in most crop plants results in natural gene containment. Chloroplast transformation also eliminates positional effects that are frequently observed with nuclear transformation and no gene silencing has been observed so far at the level of transcription or translation. Consequently, independent chloroplast transgenic lines have very similar levels of foreign gene expression and there is no need to screen hundreds of transgenic events. The chloroplast genome has also been used in molecular farming to express human therapeutic proteins, vaccines for human or animal use and biomaterials. In this study we have produced a Nicotiana tabacum cv. petit Havana chloroplast transgenic line that expresses a cholera toxin B subunit (from Vibrio Cholerae)-human proinsulin (a,b and c chain) fusion protein, designated CTB-Pris. The pLD-PW vector contains the CTB-Pris gene cloned into the universal chloroplast transformation vector pLD-ctv in which the 16S rRNA promoter drives the aadA gene selectable marker, which confers resistance to spectinomycin; the psbA 5' untranslated region (UTR) which enhances translation of CTB-Pris in the presence of light and the psbA 3'UTR confers transcript stability. The trnI and trnA homologous flanking sequences facilitated site-specific integration of transgenes into the tobacco chloroplast genome. Site-specific integration was demonstrated by PCR and Southern blot analysis with probes for CTB-Pris. Western Blot analysis has demonstrated the presence of abundant CTB-Pris in transgenic plants with both CTB polyclonal and proinsulin monoclonal antibodies. Southern blot analysis has also confirmed that homoplasmy had been achieved in the T0 generation. The expression levels for CTB-Proinsulin varied between 270ìg/100mg to 364.8ìg/100mg of plant tissue which equates to ~30% total soluble protein. In the second study the E. coli ubiC gene that codes for chorismate pyruvate-lyase (CPL) was integrated in the tobacco chloroplast genome under the control of the light-regulated psbA 5' untranslated region. CPL catalyzes the direct conversion of chorismate – an important branch point intermediate in the shikimate pathway that is exclusively synthesized in plastids – to pHBA and pyruvate. pHBA is the major monomer in liquid crystal polymers (LCPs). These thermotropic polyesters have excellent properties, including high strength/stiffness, low melt viscosity, property retention at elevated temperatures, environmental resistance and low gas permeability. The leaf content of pHBA glucose conjugates in fully mature T1 plants exposed to continuous light (total pooled material) varied between 13-18% DW, while the oldest leaves had levels as high as 26.5% DW. The highest CPL enzyme activity observed in total leaf material was 50,783 pkat/mg of protein, which is equivalent to ~35% of the total soluble protein. Animal studies in the Daniell lab, suggest that the CTB-Proinsulin producing plants suppress insulitis and clinical symptoms of diabetes in NOD mice. These observations demonstrate the versatility of chloroplast gene expression for production of biopharmaceuticals and biopolymers.
Show less - Date Issued
- 2006
- Identifier
- CFE0001056, ucf:46794
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0001056
- Title
- Expression and functional evaluation of exendin 4 fused to cholera toxin B subunit in tobacco chloroplasts to treat type 2 diabetes.
- Creator
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Nityanandam, Ramya, Daniell, Henry, Naser, Saleh, Siddiqi, Shadab, University of Central Florida
- Abstract / Description
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The prevalence of type 2 diabetes has been steadily increasing around the globe. Glucagon like peptide (GLP-1), a powerful incretin increases insulin secretion in a glucose dependent manner. But GLP-1 is subjected to rapid enzymatic degradation (half-life: 2 min in circulation). The commercially available GLP-1 analog, exenatide has a longer half life with potent insulinotropic effects (about 2.4 hr) which requires cold storage and daily subcutaneous injections. In this study, exendin 4 (EX4)...
Show moreThe prevalence of type 2 diabetes has been steadily increasing around the globe. Glucagon like peptide (GLP-1), a powerful incretin increases insulin secretion in a glucose dependent manner. But GLP-1 is subjected to rapid enzymatic degradation (half-life: 2 min in circulation). The commercially available GLP-1 analog, exenatide has a longer half life with potent insulinotropic effects (about 2.4 hr) which requires cold storage and daily subcutaneous injections. In this study, exendin 4 (EX4), lizard derived GLP-1R agonist, was expressed as cholera toxin B subunit (CTB)-fusion protein in chloroplasts of tobacco to facilitate transmucosal delivery in the gut by utilizing the ability of CTB pentamer to bind the GM1 receptors on the intestinal epithelium and to bioencapsulate EX4 within plant cells to confer protection in the digestive system. The LAMD tobacco leaves were bombarded with chloroplast vectors expressing modified EX4. The transgene integration was confirmed by PCR analysis and Southern blot analysis. Densitometric analysis revealed expression level of the protein varied from 9-13% of the total leaf protein depending on the developmental stage and time of harvest. The pentameric structure and functionality of CTB-EX4 fusion protein was confirmed by CTB-GM1 binding assay. The effect of transplastomic protein on insulin secretion was tested in ?-TC6, a mouse pancreatic cell line. The plant derived CTB-EX4, partially purified with anti-CTB antibody conjugated protein A beads, showed the increase of insulin ~ 2.5 fold increase when compared to untreated cells. The transplastomic protein showed a linear increase in insulin secretion comparable to the commercially available EX4. The current cost of treatment with EX4 varies between $1800-$2200, annually. Production of functional EX4 in plants should facilitate low cost orally deliverable form of this drug for treatment of type 2 diabetes.
Show less - Date Issued
- 2011
- Identifier
- CFE0004485, ucf:49306
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004485
- Title
- AMYLOID-BETA42 TOXICITY REDUCTION IN HUMAN NEUROBLASTOMA CELLS USING CHOLERA TOXIN B SUBUNIT-MYELIN BASIC PROTEIN EXPRESSED IN CHLOROPLASTS.
- Creator
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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.
Show less - Date Issued
- 2012
- Identifier
- CFH0004249, ucf:44916
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH0004249
