Development of New Platforms for Efficient Production and Purification of Recombinant Protein in Higher Plants

The world-wide demand for recombinant proteins continuously increases as new medical and industrial applications are developed. Higher plants have the potential to help meet this rising demand as green bioreactors. A major hurdle, however, is low recombinant protein yields in higher plants and, as with many production systems, high cost associated with downstream purification and the production of short peptides. The goal of this research was to address each of these problems to further increase the utility of plant bioreactors. The short antigenic peptide 277 (p277) from heat shock protein 60, which has the potential for use in the prevention of type 1 diabetes, was fused to an known adjuvant, the non-toxic B subunit of cholera toxin (CTB). The fused CTB-p277 retained the ability to form a homo-tetramer, and bound to GM1 ganglioside, allowing for the oral delivery of the fusion protein to induce mucosal tolerance and prevention of diabetes. Protein yield increases can be achieved by either increasing overall plant yield or increasing the relative accumulation of the desired protein. Using RNA interference, a knockdown of Solanum tuberosum plastidic ATP/ADP transporter, involved in energy transport into heterotrophic plastids, resulted in a 30% increase in tuber biomass, as well as a two-fold increase in soluble protein content. In addition, expression of a monoclonal antibody in the knockdown line produced double the concentration of antibody per soluble protein compared to wild-type. Taken together, this is a 4-fold yield increase compared to wild-type. Protein purification accounts for a significant portion of its production cost. We developed the recombinant production of soybean agglutinin (SBA) for potential use as an affinity tag. SBA was purified to high quality using an agarose-N-acetyl-D-galactosamine column, resulting in a 1-step purification process. Recombinant SBA performed identically to native SBA during in vitro assays, including agglutination of red blood cells (RBC). A fusion of SBA with green fluorescent protein (GFP) resulted in SBA-GFP that retained its in vivo fluorescence, purification through a 1-step process as well as the ability to agglutinate RBC and in vitro fluorescence of the agglutinated cells

The maize INDETERMINATE1 flowering time regulator defines a highly conserved zinc finger protein family in higher plants

BACKGROUND: The maize INDETERMINATE1 gene, ID1, is a key regulator of the transition to flowering and the founding member of a transcription factor gene family that encodes a protein with a distinct arrangement of zinc finger motifs. The zinc fingers and surrounding sequence make up the signature ID domain (IDD), which appears to be found in all higher plant genomes. The presence of zinc finger domains and previous biochemical studies showing that ID1 binds to DNA suggests that members of this gene family are involved in transcriptional regulation. RESULTS: Comparison of IDD genes identified in Arabidopsis and rice genomes, and all IDD genes discovered in maize EST and genomic databases, suggest that ID1 is a unique member of this gene family. High levels of sequence similarity amongst all IDD genes from maize, rice and Arabidopsis suggest that they are derived from a common ancestor. Several unique features of ID1 suggest that it is a divergent member of the maize IDD family. Although no clear ID1 ortholog was identified in the Arabidopsis genome, highly similar genes that encode proteins with identity extending beyond the ID domain were isolated from rice and sorghum. Phylogenetic comparisons show that these putative orthologs, along with maize ID1, form a group separate from other IDD genes. In contrast to ID1 mRNA, which is detected exclusively in immature leaves, several maize IDD genes showed a broad range of expression in various tissues. Further, Western analysis with an antibody that cross-reacts with ID1 protein and potential orthologs from rice and sorghum shows that all three proteins are detected in immature leaves only. CONCLUSION: Comparative genomic analysis shows that the IDD zinc finger family is highly conserved among both monocots and dicots. The leaf-specific ID1 expression pattern distinguishes it from other maize IDD genes examined. A similar leaf-specific localization pattern was observed for the putative ID1 protein orthologs from rice and sorghum. These similarities between ID1 and closely related genes in other grasses point to possible similarities in function

A novel expression platform for the production of diabetes-associated autoantigen human glutamic acid decarboxylase (hGAD65)

<p>Abstract</p> <p>Background</p> <p>Human glutamic acid decarboxylase 65 (hGAD65) is a key autoantigen in type 1 diabetes, having much potential as an important marker for the prediction and diagnosis of type 1 diabetes, and for the development of novel antigen-specific therapies for the treatment of type 1 diabetes. However, recombinant production of hGAD65 using conventional bacterial or mammalian cell culture-based expression systems or nuclear transformed plants is limited by low yield and low efficiency. Chloroplast transformation of the unicellular eukaryotic alga <it>Chlamydomonas reinhardtii </it>may offer a potential solution.</p> <p>Results</p> <p>A DNA cassette encoding full-length <it>hGAD65</it>, under the control of the <it>C. reinhardtii </it>chloroplast <it>rbc</it>L promoter and 5'- and 3'-UTRs, was constructed and introduced into the chloroplast genome of <it>C. reinhardtii </it>by particle bombardment. Integration of <it>hGAD65 </it>DNA into the algal chloroplast genome was confirmed by PCR. Transcriptional expression of <it>hGAD65 </it>was demonstrated by RT-PCR. Immunoblotting verified the expression and accumulation of the recombinant protein. The antigenicity of algal-derived hGAD65 was demonstrated with its immunoreactivity to diabetic sera by ELISA and by its ability to induce proliferation of spleen cells from NOD mice. Recombinant hGAD65 accumulated in transgenic algae, accounts for approximately 0.25–0.3% of its total soluble protein.</p> <p>Conclusion</p> <p>Our results demonstrate the potential value of <it>C. reinhardtii </it>chloroplasts as a novel platform for rapid mass production of immunologically active hGAD65. This demonstration opens the future possibility for using algal chloroplasts as novel bioreactors for the production of many other biologically active mammalian therapeutic proteins.</p

Altered gravitropic response, amyloplast sedimentation and circumutation in the Arabidopsis shoot gravitropism 5 mutant are associated with reduced starch levels

Abstract Plants have developed sophisticated gravity sensing mechanisms to interpret environmental signals that are vital for optimum plant growth. Loss of SHOOT GRAVITROPISM 5 (SGR5) gene function has been shown to affect the gravitropic response of Arabidopsis inflorescence stems. SGR5 is a member of the INDETERMINATE DOMAIN (IDD) zinc finger protein family of putative transcription factors. As part of an ongoing functional analysis of Arabidopsis IDD genes (AtIDD) we have extended the characterisation of SGR5, and show that gravity sensing amyloplasts in the shoot endodermis of sgr5 mutants sediment more slowly than wild type, suggesting a defect in gravity perception. This is correlated with lower amyloplast starch levels, which may account for the reduced gravitropic sensitivity in sgr5. Further, we find that sgr5 mutants have a severely attenuated stem circumnutation movement typified by a reduced amplitude and an decreased periodicity. adg1-1 and sex1-1 mutants, which contain no starch or increased starch, respectively, also show alterations in the amplitude and period of circumnutation. Together these results suggest that plant growth movement may depend on starch levels and/or gravity sensing. Overall, we propose that loss of SGR5 regulatory activity affects starch accumulation in Arabidopsis shoot tissues and causes decreased sensitivity to gravity and diminished circumnutational movements