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-specifi...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
BMC
2008-11-01
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| Series: | BMC Biotechnology |
| Online Access: | http://www.biomedcentral.com/1472-6750/8/87 |
| _version_ | 1818346499629645824 |
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| author | Maxwell Denis Tremblay Reynald Brandsma Martin Wang Xiaofeng Jevnikar Anthony M Huner Norm Ma Shengwu |
| author_facet | Maxwell Denis Tremblay Reynald Brandsma Martin Wang Xiaofeng Jevnikar Anthony M Huner Norm Ma Shengwu |
| author_sort | Maxwell Denis |
| collection | DOAJ |
| description | <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> |
| first_indexed | 2024-12-13T17:19:15Z |
| format | Article |
| id | doaj.art-8a14d704500345698a69a0bf1ce11386 |
| institution | Directory Open Access Journal |
| issn | 1472-6750 |
| language | English |
| last_indexed | 2024-12-13T17:19:15Z |
| publishDate | 2008-11-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Biotechnology |
| spelling | doaj.art-8a14d704500345698a69a0bf1ce113862022-12-21T23:37:21ZengBMCBMC Biotechnology1472-67502008-11-01818710.1186/1472-6750-8-87A novel expression platform for the production of diabetes-associated autoantigen human glutamic acid decarboxylase (hGAD65)Maxwell DenisTremblay ReynaldBrandsma MartinWang XiaofengJevnikar Anthony MHuner NormMa Shengwu<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>http://www.biomedcentral.com/1472-6750/8/87 |
| spellingShingle | Maxwell Denis Tremblay Reynald Brandsma Martin Wang Xiaofeng Jevnikar Anthony M Huner Norm Ma Shengwu A novel expression platform for the production of diabetes-associated autoantigen human glutamic acid decarboxylase (hGAD65) BMC Biotechnology |
| title | A novel expression platform for the production of diabetes-associated autoantigen human glutamic acid decarboxylase (hGAD65) |
| title_full | A novel expression platform for the production of diabetes-associated autoantigen human glutamic acid decarboxylase (hGAD65) |
| title_fullStr | A novel expression platform for the production of diabetes-associated autoantigen human glutamic acid decarboxylase (hGAD65) |
| title_full_unstemmed | A novel expression platform for the production of diabetes-associated autoantigen human glutamic acid decarboxylase (hGAD65) |
| title_short | A novel expression platform for the production of diabetes-associated autoantigen human glutamic acid decarboxylase (hGAD65) |
| title_sort | novel expression platform for the production of diabetes associated autoantigen human glutamic acid decarboxylase hgad65 |
| url | http://www.biomedcentral.com/1472-6750/8/87 |
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