Current Search: Chloroplast biotechnology (x)
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- Title
- LOW COST PRODUCTION OF PROINSULIN IN TOBACCO AND LETTUCE CHLOROPLASTS FOR INJECTABLE OR ORAL DELIVERY OF FUNCTIONAL INSULIN AND C-PEPTIDE.
- Creator
-
Burberry, Diane, Daniell, Henry, University of Central Florida
- Abstract / Description
-
Current treatment for type I diabetes includes delivery of insulin via injection or pump, which is highly invasive and expensive. The production of chloroplast-derived proinsulin should reduce cost and facilitate oral delivery. Therefore, tobacco and lettuce chloroplasts were transformed with the cholera toxin B subunit fused with human proinsulin (A, B, and C peptides) containing three furin cleavage sites (CTB-PFx3). Transplastomic lines were confirmed for site-specific integration of...
Show moreCurrent treatment for type I diabetes includes delivery of insulin via injection or pump, which is highly invasive and expensive. The production of chloroplast-derived proinsulin should reduce cost and facilitate oral delivery. Therefore, tobacco and lettuce chloroplasts were transformed with the cholera toxin B subunit fused with human proinsulin (A, B, and C peptides) containing three furin cleavage sites (CTB-PFx3). Transplastomic lines were confirmed for site-specific integration of transgene and homoplasmy. Old tobacco leaves accumulated proinsulin up to 47% of total leaf protein (TLP). Old lettuce leaves accumulated proinsulin up to 53% TLP. Accumulation was so stable that up to ~40% proinsulin in TLP was observed even in senescent and dried lettuce leaves, facilitating their processing and storage in the field. Based on the yield of only monomers and dimers of proinsulin (3 mg/g leaf, a significant underestimation), with a 50% loss of protein during the purification process, one acre of tobacco could yield up to 20 million daily doses of insulin per year. Proinsulin from tobacco leaves was purified up to 98% using metal affinity chromatography without any His-tag. Furin protease cleaved insulin peptides in vitro. Oral delivery of unprocessed proinsulin bioencapsulated in plant cells or injectable delivery into mice showed reduction in blood glucose levels similar to processed commercial insulin. C-peptide should aid in longterm treatment of diabetic complications including stimulation of nerve and renal functions. Hyper-expression of functional proinsulin and exceptional stability in dehydrated leaves offer a low cost platform for oral and injectable delivery of cleavable proinsulin.
Show less - Date Issued
- 2010
- Identifier
- CFE0003257, ucf:48554
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0003257
- Title
- Expression of Lipase from Mycobacterium tuberculosis in Nicotiana tobacum and Lactuca sativa Chloroplasts.
- Creator
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Lloyd, Bethany, Daniell, Henry, Kolattukudy, Pappachan, Self, William, University of Central Florida
- Abstract / Description
-
Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis (M. tuberculosis), is a global threat and the leading cause of death among individuals infected with HIV. TB treatment requires multi-drug cocktails, due to the increasing rates of drug resistance of the bacterium. With multi-drug cocktails, strains have been documented to be resistant to all major drugs in the fight against TB. Since the strains are drug resistant, it calls for an increasing need for vaccine and treatment...
Show moreTuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis (M. tuberculosis), is a global threat and the leading cause of death among individuals infected with HIV. TB treatment requires multi-drug cocktails, due to the increasing rates of drug resistance of the bacterium. With multi-drug cocktails, strains have been documented to be resistant to all major drugs in the fight against TB. Since the strains are drug resistant, it calls for an increasing need for vaccine and treatment development for the purpose of preventing and managing the disease. The most widely distributed vaccine against TB is Bacillus Calmette-Gue(&)#180;rin (BCG). Apart from being ineffective in certain individuals, BCG offers only a limited timeframe of protection, is unable to serve as a booster for extending this timeframe and due to the intradermal route of administration requires costly refrigeration and syringes.LipY protein, a M. tuberculosis cell wall lipase, may play a potential role as not only a drug target but a potential vaccine antigen. LipY is known to be up-regulated during both active infection and dormancy. In a previous study, sera from TB patients had shown an IgG and IgM response against it. In this study transplastomic Lactuca sativa and Nicotiana tabacum plants were generated by transforming the chloroplasts through the particle delivery system with pLsDv-LipY and pLD-LipY vectors respectively. The vectors were flanked by the native trnI and trnA gene sequence to facilitate homologous recombination into the chloroplast genome. The vector also contained the 16S rRNA promoter, the selectable marker gene, aadA for specitinomycin resistance, the rbcL untranslated region, the LsPpsbA (PpsbA in N. tabacum) promoter, and LsTpsbA (tpsbA in N. tabacum) untranslated region. Site specific integration of the LipY gene into the chloroplast genome was confirmed by PCR. Homoplasmy of transplastomic plants was confirmed by Southern blot analysis. These plants showed normal growth and were fertile, producing seeds. Once germinated, these seeds did not show Mendelian segregation of the transgene. Immunoblot analysis was performed to analyze the expression of the LipY protein. A 40kDa protein was produced in E.coli, and a 25kDa protein was produced in chloroplasts; a cleaved product in chloroplasts is still valuable as an antigen for vaccine production. Future studies will include testing this chloroplast derived antigen in animal models for vaccine development. ?
Show less - Date Issued
- 2012
- Identifier
- CFE0004502, ucf:49289
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004502
