Successful production of recombinant buckwheat cysteine-rich aspartic protease in Escherichia coli
Herein, the expression of recombinant cysteine-rich atypical buckwheat (Fagopyrum esculentum) aspartic protease (FeAPL1) in five Escherichia coli strains differing in their expression capabilities is presented. It was shown that the expression success depended highly on the choice of FeAPL1 fusion p...
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| Format: | Article |
| Language: | English |
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Serbian Chemical Society
2009-01-01
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| Series: | Journal of the Serbian Chemical Society |
| Subjects: | |
| Online Access: | http://www.doiserbia.nb.rs/img/doi/0352-5139/2009/0352-51390906607M.pdf |
| _version_ | 1818330261257977856 |
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| author | Milisavljević Mira D. Papić Dražen R. Timotijević Gordana S. Maksimović Vesna R. |
| author_facet | Milisavljević Mira D. Papić Dražen R. Timotijević Gordana S. Maksimović Vesna R. |
| author_sort | Milisavljević Mira D. |
| collection | DOAJ |
| description | Herein, the expression of recombinant cysteine-rich atypical buckwheat (Fagopyrum esculentum) aspartic protease (FeAPL1) in five Escherichia coli strains differing in their expression capabilities is presented. It was shown that the expression success depended highly on the choice of FeAPL1 fusion partner. His6-FeAPL1 was produced in large quantities as an insoluble protein localized in inclusion bodies. On the other hand, MBP-FeAPL1 was localized in both the cytoplasm and inclusion bodies in BL21 and Rosetta-gami strains. Only purified soluble MBP-FeAPL1 from Rosetta-gami cells showed proteolytic activity at pH 3.0 with BSA as the substrate. The results also indicated that FeAPL1 contained a PRO segment that had to be removed for the enzyme activity to appear. The activity of FeAPL1 produced in the Rosetta-gami strain, which enables disulfide bond formation, indicated the importance of the twelve cysteine residues for correct folding and functionality. |
| first_indexed | 2024-12-13T13:01:08Z |
| format | Article |
| id | doaj.art-4908068b01b84df1991419b60326f555 |
| institution | Directory Open Access Journal |
| issn | 0352-5139 1820-7421 |
| language | English |
| last_indexed | 2024-12-13T13:01:08Z |
| publishDate | 2009-01-01 |
| publisher | Serbian Chemical Society |
| record_format | Article |
| series | Journal of the Serbian Chemical Society |
| spelling | doaj.art-4908068b01b84df1991419b60326f5552022-12-21T23:45:01ZengSerbian Chemical SocietyJournal of the Serbian Chemical Society0352-51391820-74212009-01-0174660761810.2298/JSC0906607M0352-51390906607MSuccessful production of recombinant buckwheat cysteine-rich aspartic protease in Escherichia coliMilisavljević Mira D.0Papić Dražen R.1Timotijević Gordana S.2Maksimović Vesna R.3Institut za molekularnu genetiku i genetičko inženjerstvo, BeogradInstitut za molekularnu genetiku i genetičko inženjerstvo, BeogradInstitut za molekularnu genetiku i genetičko inženjerstvo, BeogradInstitut za molekularnu genetiku i genetičko inženjerstvo, BeogradHerein, the expression of recombinant cysteine-rich atypical buckwheat (Fagopyrum esculentum) aspartic protease (FeAPL1) in five Escherichia coli strains differing in their expression capabilities is presented. It was shown that the expression success depended highly on the choice of FeAPL1 fusion partner. His6-FeAPL1 was produced in large quantities as an insoluble protein localized in inclusion bodies. On the other hand, MBP-FeAPL1 was localized in both the cytoplasm and inclusion bodies in BL21 and Rosetta-gami strains. Only purified soluble MBP-FeAPL1 from Rosetta-gami cells showed proteolytic activity at pH 3.0 with BSA as the substrate. The results also indicated that FeAPL1 contained a PRO segment that had to be removed for the enzyme activity to appear. The activity of FeAPL1 produced in the Rosetta-gami strain, which enables disulfide bond formation, indicated the importance of the twelve cysteine residues for correct folding and functionality.http://www.doiserbia.nb.rs/img/doi/0352-5139/2009/0352-51390906607M.pdfaspartic proteasehis taginclusion bodiesmbprecombinant protein |
| spellingShingle | Milisavljević Mira D. Papić Dražen R. Timotijević Gordana S. Maksimović Vesna R. Successful production of recombinant buckwheat cysteine-rich aspartic protease in Escherichia coli Journal of the Serbian Chemical Society aspartic protease his tag inclusion bodies mbp recombinant protein |
| title | Successful production of recombinant buckwheat cysteine-rich aspartic protease in Escherichia coli |
| title_full | Successful production of recombinant buckwheat cysteine-rich aspartic protease in Escherichia coli |
| title_fullStr | Successful production of recombinant buckwheat cysteine-rich aspartic protease in Escherichia coli |
| title_full_unstemmed | Successful production of recombinant buckwheat cysteine-rich aspartic protease in Escherichia coli |
| title_short | Successful production of recombinant buckwheat cysteine-rich aspartic protease in Escherichia coli |
| title_sort | successful production of recombinant buckwheat cysteine rich aspartic protease in escherichia coli |
| topic | aspartic protease his tag inclusion bodies mbp recombinant protein |
| url | http://www.doiserbia.nb.rs/img/doi/0352-5139/2009/0352-51390906607M.pdf |
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