Expression, Purification and Characterization of Functional Teduglutide Using GST Fusion System in Prokaryotic Cells
Purpose: Teduglutide is the first and only FDA-approved drug for long-term treatment of short bowel syndrome (SBS). The current study aimed to present an approach for production of teduglutide using recombinant DNA technology. Methods: The coding gene for teduglutide was cloned into pGEX-2T vector,...
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| Format: | Article |
| Language: | English |
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Tabriz University of Medical Sciences
2023-07-01
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| Series: | Advanced Pharmaceutical Bulletin |
| Subjects: | |
| Online Access: | https://apb.tbzmed.ac.ir/PDF/apb-13-592.pdf |
| _version_ | 1797673436958425088 |
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| author | Ali Akbar Alizadeh Saba Rasouli Omid Jamshidi Kandjani Salar Hemmati Siavoush Dastmalchi |
| author_facet | Ali Akbar Alizadeh Saba Rasouli Omid Jamshidi Kandjani Salar Hemmati Siavoush Dastmalchi |
| author_sort | Ali Akbar Alizadeh |
| collection | DOAJ |
| description | Purpose: Teduglutide is the first and only FDA-approved drug for long-term treatment of short bowel syndrome (SBS). The current study aimed to present an approach for production of teduglutide using recombinant DNA technology. Methods: The coding gene for teduglutide was cloned into pGEX-2T vector, where coding sequence for factor Xa cleavage site was added between GST and teduglutide coding genes. The GST-teduglutide protein was overexpressed in E. coli BL21 (DE3) strain and affinity purified using glutathione sepharose affinity column. Results: On-column proteolytic activity of factor Xa followed by size exclusion chromatography resulted in the pure teduglutide. Circular dichroism (CD) spectropolarimetry showed that the produced teduglutide folds into mainly α-helical structure (>50%), as expected. In mass spectroscopy analysis, the fragments of teduglutide resulted by cyanogen bromide cleavage as well as those expected theoretically due to mass fragmentation were identified. The functionality of the produced peptide was evaluated by measuring its proliferative effect on Caco2 intestinal epithelial cells, and the results indicated that produced teduglutide induces cell proliferation by 19±0.30 and 33±7.82 % at 1.21 and 3.64 µM concentrations, respectively, compared to untreated cells. Conclusion: Teduglutide was successfully expressed and purified and its functionality and structural integrity were confirmed by in vitro experiments. We believe that the experimental-scale method presented in the current study can be useful for pilot-scale and also industrial-scale production of teduglutide. |
| first_indexed | 2024-03-11T21:44:31Z |
| format | Article |
| id | doaj.art-55ae25c3538d41cbb7d4172498f7ec70 |
| institution | Directory Open Access Journal |
| issn | 2228-5881 2251-7308 |
| language | English |
| last_indexed | 2024-03-11T21:44:31Z |
| publishDate | 2023-07-01 |
| publisher | Tabriz University of Medical Sciences |
| record_format | Article |
| series | Advanced Pharmaceutical Bulletin |
| spelling | doaj.art-55ae25c3538d41cbb7d4172498f7ec702023-09-26T10:45:47ZengTabriz University of Medical SciencesAdvanced Pharmaceutical Bulletin2228-58812251-73082023-07-0113359260010.34172/apb.2023.064apb-39033Expression, Purification and Characterization of Functional Teduglutide Using GST Fusion System in Prokaryotic CellsAli Akbar Alizadeh0Saba Rasouli1Omid Jamshidi Kandjani2Salar Hemmati3Siavoush Dastmalchi4Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.Pharmaceutical Analysis Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.Purpose: Teduglutide is the first and only FDA-approved drug for long-term treatment of short bowel syndrome (SBS). The current study aimed to present an approach for production of teduglutide using recombinant DNA technology. Methods: The coding gene for teduglutide was cloned into pGEX-2T vector, where coding sequence for factor Xa cleavage site was added between GST and teduglutide coding genes. The GST-teduglutide protein was overexpressed in E. coli BL21 (DE3) strain and affinity purified using glutathione sepharose affinity column. Results: On-column proteolytic activity of factor Xa followed by size exclusion chromatography resulted in the pure teduglutide. Circular dichroism (CD) spectropolarimetry showed that the produced teduglutide folds into mainly α-helical structure (>50%), as expected. In mass spectroscopy analysis, the fragments of teduglutide resulted by cyanogen bromide cleavage as well as those expected theoretically due to mass fragmentation were identified. The functionality of the produced peptide was evaluated by measuring its proliferative effect on Caco2 intestinal epithelial cells, and the results indicated that produced teduglutide induces cell proliferation by 19±0.30 and 33±7.82 % at 1.21 and 3.64 µM concentrations, respectively, compared to untreated cells. Conclusion: Teduglutide was successfully expressed and purified and its functionality and structural integrity were confirmed by in vitro experiments. We believe that the experimental-scale method presented in the current study can be useful for pilot-scale and also industrial-scale production of teduglutide.https://apb.tbzmed.ac.ir/PDF/apb-13-592.pdfrecombinant technologypeptideteduglutideaffinity chromatographysbssize exclusion chromatography |
| spellingShingle | Ali Akbar Alizadeh Saba Rasouli Omid Jamshidi Kandjani Salar Hemmati Siavoush Dastmalchi Expression, Purification and Characterization of Functional Teduglutide Using GST Fusion System in Prokaryotic Cells Advanced Pharmaceutical Bulletin recombinant technology peptide teduglutide affinity chromatography sbs size exclusion chromatography |
| title | Expression, Purification and Characterization of Functional Teduglutide Using GST Fusion System in Prokaryotic Cells |
| title_full | Expression, Purification and Characterization of Functional Teduglutide Using GST Fusion System in Prokaryotic Cells |
| title_fullStr | Expression, Purification and Characterization of Functional Teduglutide Using GST Fusion System in Prokaryotic Cells |
| title_full_unstemmed | Expression, Purification and Characterization of Functional Teduglutide Using GST Fusion System in Prokaryotic Cells |
| title_short | Expression, Purification and Characterization of Functional Teduglutide Using GST Fusion System in Prokaryotic Cells |
| title_sort | expression purification and characterization of functional teduglutide using gst fusion system in prokaryotic cells |
| topic | recombinant technology peptide teduglutide affinity chromatography sbs size exclusion chromatography |
| url | https://apb.tbzmed.ac.ir/PDF/apb-13-592.pdf |
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