High-level expression and rapid purification of rare-codon genes from hyperthermophilic archaea by the GST gene fusion system.
In this study, we compared two gene fusion expression strategies using two rare codon genes (Ssh10b and MtGrxM) from archaea as a model system. Both genes can be highly expressed as N- or C-terminal fusion partners to GST or the intein/chitin-binding tag. However, the fusion protein with intein tag...
| Main Authors: | , |
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| Format: | Journal article |
| Language: | English |
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2003
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| _version_ | 1826256501074821120 |
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| author | Wu, X Oppermann, U |
| author_facet | Wu, X Oppermann, U |
| author_sort | Wu, X |
| collection | OXFORD |
| description | In this study, we compared two gene fusion expression strategies using two rare codon genes (Ssh10b and MtGrxM) from archaea as a model system. Both genes can be highly expressed as N- or C-terminal fusion partners to GST or the intein/chitin-binding tag. However, the fusion protein with intein tag could not be cleaved, even under stringent conditions, possibly due to steric hindrance, thus preventing further purification. In contrast, the GST fusion system could increase protein expression level and the corresponding fusion protein could be easily cleaved by thrombin. After binding to glutathione sepharose, the fusion protein was cleaved on column, and a roughly purified protein fraction was eluted. This fraction was purified by heating at 80 degrees C for 10 min, followed by centrifugation. The correct total mass and N-terminal primary structure were confirmed by mass spectrometry and Edman degradation. Both constructs were used for in vitro expression, and similar results were obtained, indicating higher expression levels of the GST tag vs. intein/chitin tag. Taken together, our results suggest that the GST fusion system can be used as a considerable alternative to synthetic genes for the expression of rare codon genes. The affinity chromatography purification followed by a heating step is an efficient and convenient method for thermostable protein purification. |
| first_indexed | 2024-03-06T18:03:12Z |
| format | Journal article |
| id | oxford-uuid:008797ee-951d-45c9-bb63-1031d2a4d651 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-06T18:03:12Z |
| publishDate | 2003 |
| record_format | dspace |
| spelling | oxford-uuid:008797ee-951d-45c9-bb63-1031d2a4d6512022-03-26T08:29:56ZHigh-level expression and rapid purification of rare-codon genes from hyperthermophilic archaea by the GST gene fusion system.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:008797ee-951d-45c9-bb63-1031d2a4d651EnglishSymplectic Elements at Oxford2003Wu, XOppermann, UIn this study, we compared two gene fusion expression strategies using two rare codon genes (Ssh10b and MtGrxM) from archaea as a model system. Both genes can be highly expressed as N- or C-terminal fusion partners to GST or the intein/chitin-binding tag. However, the fusion protein with intein tag could not be cleaved, even under stringent conditions, possibly due to steric hindrance, thus preventing further purification. In contrast, the GST fusion system could increase protein expression level and the corresponding fusion protein could be easily cleaved by thrombin. After binding to glutathione sepharose, the fusion protein was cleaved on column, and a roughly purified protein fraction was eluted. This fraction was purified by heating at 80 degrees C for 10 min, followed by centrifugation. The correct total mass and N-terminal primary structure were confirmed by mass spectrometry and Edman degradation. Both constructs were used for in vitro expression, and similar results were obtained, indicating higher expression levels of the GST tag vs. intein/chitin tag. Taken together, our results suggest that the GST fusion system can be used as a considerable alternative to synthetic genes for the expression of rare codon genes. The affinity chromatography purification followed by a heating step is an efficient and convenient method for thermostable protein purification. |
| spellingShingle | Wu, X Oppermann, U High-level expression and rapid purification of rare-codon genes from hyperthermophilic archaea by the GST gene fusion system. |
| title | High-level expression and rapid purification of rare-codon genes from hyperthermophilic archaea by the GST gene fusion system. |
| title_full | High-level expression and rapid purification of rare-codon genes from hyperthermophilic archaea by the GST gene fusion system. |
| title_fullStr | High-level expression and rapid purification of rare-codon genes from hyperthermophilic archaea by the GST gene fusion system. |
| title_full_unstemmed | High-level expression and rapid purification of rare-codon genes from hyperthermophilic archaea by the GST gene fusion system. |
| title_short | High-level expression and rapid purification of rare-codon genes from hyperthermophilic archaea by the GST gene fusion system. |
| title_sort | high level expression and rapid purification of rare codon genes from hyperthermophilic archaea by the gst gene fusion system |
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