An effective extracellular protein secretion by an ABC transporter system in Escherichia coli: statistical modeling and optimization of cyclodextrin glucanotransferase secretory production
Direct transport of recombinant protein from cytosol to extracellular medium offers great advantages, such as high specific activity and a simple purification step. This work presents an investigation on the potential of an ABC (ATP-binding cassette) transporter system, the hemolysin transport syste...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Springer-Verlag
2011
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| Online Access: | http://psasir.upm.edu.my/id/eprint/22354/1/An%20effective%20extracellular%20protein%20secretion%20by%20an%20ABC%20transporter%20system%20in%20Escherichia%20coli.pdf |
| _version_ | 1796970144891666432 |
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| author | Kheng, Oon Low Mahadi, Nor Muhammad Abdul Rahim, Raha Rabu, Amir Abu Bakar, Farah Diba Abdul Murad, Abdul Munir Md. Illias, Rosli |
| author_facet | Kheng, Oon Low Mahadi, Nor Muhammad Abdul Rahim, Raha Rabu, Amir Abu Bakar, Farah Diba Abdul Murad, Abdul Munir Md. Illias, Rosli |
| author_sort | Kheng, Oon Low |
| collection | UPM |
| description | Direct transport of recombinant protein from cytosol to extracellular medium offers great advantages, such as high specific activity and a simple purification step. This work presents an investigation on the potential of an ABC (ATP-binding cassette) transporter system, the hemolysin transport system, for efficient protein secretion in Escherichia coli (E. coli). A higher secretory production of recombinant cyclodextrin glucanotransferase (CGTase) was achieved by a new plasmid design and subsequently by optimization of culture conditions via central composite design. An improvement of at least fourfold extracellular recombinant CGTase was obtained using the new plasmid design. The optimization process consisted of 20 experiments involving six star points and six replicates at the central point. The predicted optimum culture conditions for maximum recombinant CGTase secretion were found to be 25.76 μM IPTG, 1.0% (w/v) arabinose and 34.7°C post-induction temperature, with a predicted extracellular CGTase activity of 68.76 U/ml. Validation of the model gave an extracellular CGTase activity of 69.15 ± 0.71 U/ml, resulting in a 3.45-fold increase compared to the initial conditions. This corresponded to an extracellular CGTase yield of about 0.58 mg/l. We showed that a synergistic balance of transported protein and secretory pathway is important for efficient protein transport. In addition, we also demonstrated the first successful removal of the C-terminal secretion signal from the transported fusion protein by thrombin proteolytic cleavage. |
| first_indexed | 2024-03-06T07:53:42Z |
| format | Article |
| id | upm.eprints-22354 |
| institution | Universiti Putra Malaysia |
| language | English |
| last_indexed | 2024-03-06T07:53:42Z |
| publishDate | 2011 |
| publisher | Springer-Verlag |
| record_format | dspace |
| spelling | upm.eprints-223542015-09-21T02:41:42Z http://psasir.upm.edu.my/id/eprint/22354/ An effective extracellular protein secretion by an ABC transporter system in Escherichia coli: statistical modeling and optimization of cyclodextrin glucanotransferase secretory production Kheng, Oon Low Mahadi, Nor Muhammad Abdul Rahim, Raha Rabu, Amir Abu Bakar, Farah Diba Abdul Murad, Abdul Munir Md. Illias, Rosli Direct transport of recombinant protein from cytosol to extracellular medium offers great advantages, such as high specific activity and a simple purification step. This work presents an investigation on the potential of an ABC (ATP-binding cassette) transporter system, the hemolysin transport system, for efficient protein secretion in Escherichia coli (E. coli). A higher secretory production of recombinant cyclodextrin glucanotransferase (CGTase) was achieved by a new plasmid design and subsequently by optimization of culture conditions via central composite design. An improvement of at least fourfold extracellular recombinant CGTase was obtained using the new plasmid design. The optimization process consisted of 20 experiments involving six star points and six replicates at the central point. The predicted optimum culture conditions for maximum recombinant CGTase secretion were found to be 25.76 μM IPTG, 1.0% (w/v) arabinose and 34.7°C post-induction temperature, with a predicted extracellular CGTase activity of 68.76 U/ml. Validation of the model gave an extracellular CGTase activity of 69.15 ± 0.71 U/ml, resulting in a 3.45-fold increase compared to the initial conditions. This corresponded to an extracellular CGTase yield of about 0.58 mg/l. We showed that a synergistic balance of transported protein and secretory pathway is important for efficient protein transport. In addition, we also demonstrated the first successful removal of the C-terminal secretion signal from the transported fusion protein by thrombin proteolytic cleavage. Springer-Verlag 2011-09 Article PeerReviewed application/pdf en http://psasir.upm.edu.my/id/eprint/22354/1/An%20effective%20extracellular%20protein%20secretion%20by%20an%20ABC%20transporter%20system%20in%20Escherichia%20coli.pdf Kheng, Oon Low and Mahadi, Nor Muhammad and Abdul Rahim, Raha and Rabu, Amir and Abu Bakar, Farah Diba and Abdul Murad, Abdul Munir and Md. Illias, Rosli (2011) An effective extracellular protein secretion by an ABC transporter system in Escherichia coli: statistical modeling and optimization of cyclodextrin glucanotransferase secretory production. Journal of Industrial Microbiology and Biotechnology, 38 (9). pp. 1587-1597. ISSN 1367-5435; ESSN: 1476-5535 http://link.springer.com/article/10.1007%2Fs10295-011-0949-0 10.1007/s10295-011-0949-0 |
| spellingShingle | Kheng, Oon Low Mahadi, Nor Muhammad Abdul Rahim, Raha Rabu, Amir Abu Bakar, Farah Diba Abdul Murad, Abdul Munir Md. Illias, Rosli An effective extracellular protein secretion by an ABC transporter system in Escherichia coli: statistical modeling and optimization of cyclodextrin glucanotransferase secretory production |
| title | An effective extracellular protein secretion by an ABC transporter system in Escherichia coli: statistical modeling and optimization of cyclodextrin glucanotransferase secretory production |
| title_full | An effective extracellular protein secretion by an ABC transporter system in Escherichia coli: statistical modeling and optimization of cyclodextrin glucanotransferase secretory production |
| title_fullStr | An effective extracellular protein secretion by an ABC transporter system in Escherichia coli: statistical modeling and optimization of cyclodextrin glucanotransferase secretory production |
| title_full_unstemmed | An effective extracellular protein secretion by an ABC transporter system in Escherichia coli: statistical modeling and optimization of cyclodextrin glucanotransferase secretory production |
| title_short | An effective extracellular protein secretion by an ABC transporter system in Escherichia coli: statistical modeling and optimization of cyclodextrin glucanotransferase secretory production |
| title_sort | effective extracellular protein secretion by an abc transporter system in escherichia coli statistical modeling and optimization of cyclodextrin glucanotransferase secretory production |
| url | http://psasir.upm.edu.my/id/eprint/22354/1/An%20effective%20extracellular%20protein%20secretion%20by%20an%20ABC%20transporter%20system%20in%20Escherichia%20coli.pdf |
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