Abstract

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 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 270µg/100mg to 364.8µg/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.

Notes

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Graduation Date

2006

Semester

Spring

Advisor

Daniell, Henry

Degree

Master of Science (M.S.)

College

Burnett College of Biomedical Sciences

Department

Molecular Biology and Microbiology

Format

application/pdf

Identifier

CFE0001056

URL

http://purl.fcla.edu/fcla/etd/CFE0001056

Language

English

Release Date

October 2018

Length of Campus-only Access

None

Access Status

Masters Thesis (Open Access)

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