Molecular Cloning and Production of Recombinant Phytase from Bacillus subtilis ASUIA243 in Pichia pastoris
Phytase gene obtained from Bacillus subtilis ASUIA243 was cloned into a medium vector and transformed into E. coli. Restriction enzyme digestion was conducted to get blunt-ended phytase gene and ligated into the Pichia expression vector, pPICZαA. The recombinant vector, pPICZαA-243HPp was then linea...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
IIUM Press, International Islamic University Malaysia
2011-12-01
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| Series: | International Islamic University Malaysia Engineering Journal |
| Online Access: | http://journals.iium.edu.my/ejournal/index.php/iiumej/article/view/211 |
| _version_ | 1819028565676523520 |
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| author | Nor Soleha Mohd Dali Tamrin Nuge Mohd Hafidz Mahamad Maifiah Faridah Yusof Anis Shobirin Meor Hussin Abd-Elaziem Farouk and Hamzah Mohd. Salleh |
| author_facet | Nor Soleha Mohd Dali Tamrin Nuge Mohd Hafidz Mahamad Maifiah Faridah Yusof Anis Shobirin Meor Hussin Abd-Elaziem Farouk and Hamzah Mohd. Salleh |
| author_sort | Nor Soleha Mohd Dali |
| collection | DOAJ |
| description | Phytase gene obtained from Bacillus subtilis ASUIA243 was cloned into a medium vector and transformed into E. coli. Restriction enzyme digestion was conducted to get blunt-ended phytase gene and ligated into the Pichia expression vector, pPICZαA. The recombinant vector, pPICZαA-243HPp was then linearized with PmeI and transformed into P. pastoris strain X33. Screening for multi copy gene number of transformants was done by re-plating the selected colonies on increasing concentration of zeocin. One positive clone, X243HPp#2 was then grown in BMGY media as the starting culture, followed by induction in BMMY media for protein expression study. The supernatant was then analysed by SDS-PAGE and Western blot method to check the protein expression.
ABSTRAK: Gen fitase yang didapati daripada Bacillus subtilis ASUIA243 diklonkan sebagai vektor perantara dan berubah menjadi E. coli. Sekatan pencernaan enzim dijalankan untuk mendapatkan gen fitase berhujung tumpul dan diligatkan dengan vektor ekspresi Pichia, pPICZαA. Vektor rekombinan, pPICZαA-243HPp kemudian dilinearkan dengan PmeI dan berubah menjadi P. pastoris strain X33. Penyaringan untuk nombor gen berbilang salinan yang menjalani transformasi genetik dijalankan dengan menyalur semula koloni terpilih dengan penambahan kepekatan zeocin. Satu klon positif, X243HPp#2 kemudian dibiarkan hidup dalam perantara BMGY sebagai kultur permulaan, diikuti dengan aruhan dalam perantara BMMY untuk kajian penglahiran protein. Supernatan kemudian dikaji dengan SDS-PAGE dan kaedah sap Western untuk menyemak penglahiran protein.
KEYWORDS: phytase, Bacillus subtilis, Pichia pastoris, gene cloning. |
| first_indexed | 2024-12-21T06:00:23Z |
| format | Article |
| id | doaj.art-d4b27e8d69ce478fbd5181f6c9f4da71 |
| institution | Directory Open Access Journal |
| issn | 1511-788X 2289-7860 |
| language | English |
| last_indexed | 2024-12-21T06:00:23Z |
| publishDate | 2011-12-01 |
| publisher | IIUM Press, International Islamic University Malaysia |
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| series | International Islamic University Malaysia Engineering Journal |
| spelling | doaj.art-d4b27e8d69ce478fbd5181f6c9f4da712022-12-21T19:13:46ZengIIUM Press, International Islamic University MalaysiaInternational Islamic University Malaysia Engineering Journal1511-788X2289-78602011-12-0112410.31436/iiumej.v12i4.211Molecular Cloning and Production of Recombinant Phytase from Bacillus subtilis ASUIA243 in Pichia pastorisNor Soleha Mohd Dali0Tamrin NugeMohd Hafidz Mahamad MaifiahFaridah YusofAnis Shobirin Meor HussinAbd-Elaziem Faroukand Hamzah Mohd. SallehBioprocess and Molecular Engineering Research Unit, Deaprtment of Biotechnology Engineering, International Islamic University Malaysia (IIUM), Jalan Gombak, 53100 Kuala Lumpur, Malaysia.Phytase gene obtained from Bacillus subtilis ASUIA243 was cloned into a medium vector and transformed into E. coli. Restriction enzyme digestion was conducted to get blunt-ended phytase gene and ligated into the Pichia expression vector, pPICZαA. The recombinant vector, pPICZαA-243HPp was then linearized with PmeI and transformed into P. pastoris strain X33. Screening for multi copy gene number of transformants was done by re-plating the selected colonies on increasing concentration of zeocin. One positive clone, X243HPp#2 was then grown in BMGY media as the starting culture, followed by induction in BMMY media for protein expression study. The supernatant was then analysed by SDS-PAGE and Western blot method to check the protein expression. ABSTRAK: Gen fitase yang didapati daripada Bacillus subtilis ASUIA243 diklonkan sebagai vektor perantara dan berubah menjadi E. coli. Sekatan pencernaan enzim dijalankan untuk mendapatkan gen fitase berhujung tumpul dan diligatkan dengan vektor ekspresi Pichia, pPICZαA. Vektor rekombinan, pPICZαA-243HPp kemudian dilinearkan dengan PmeI dan berubah menjadi P. pastoris strain X33. Penyaringan untuk nombor gen berbilang salinan yang menjalani transformasi genetik dijalankan dengan menyalur semula koloni terpilih dengan penambahan kepekatan zeocin. Satu klon positif, X243HPp#2 kemudian dibiarkan hidup dalam perantara BMGY sebagai kultur permulaan, diikuti dengan aruhan dalam perantara BMMY untuk kajian penglahiran protein. Supernatan kemudian dikaji dengan SDS-PAGE dan kaedah sap Western untuk menyemak penglahiran protein. KEYWORDS: phytase, Bacillus subtilis, Pichia pastoris, gene cloning.http://journals.iium.edu.my/ejournal/index.php/iiumej/article/view/211 |
| spellingShingle | Nor Soleha Mohd Dali Tamrin Nuge Mohd Hafidz Mahamad Maifiah Faridah Yusof Anis Shobirin Meor Hussin Abd-Elaziem Farouk and Hamzah Mohd. Salleh Molecular Cloning and Production of Recombinant Phytase from Bacillus subtilis ASUIA243 in Pichia pastoris International Islamic University Malaysia Engineering Journal |
| title | Molecular Cloning and Production of Recombinant Phytase from Bacillus subtilis ASUIA243 in Pichia pastoris |
| title_full | Molecular Cloning and Production of Recombinant Phytase from Bacillus subtilis ASUIA243 in Pichia pastoris |
| title_fullStr | Molecular Cloning and Production of Recombinant Phytase from Bacillus subtilis ASUIA243 in Pichia pastoris |
| title_full_unstemmed | Molecular Cloning and Production of Recombinant Phytase from Bacillus subtilis ASUIA243 in Pichia pastoris |
| title_short | Molecular Cloning and Production of Recombinant Phytase from Bacillus subtilis ASUIA243 in Pichia pastoris |
| title_sort | molecular cloning and production of recombinant phytase from bacillus subtilis asuia243 in pichia pastoris |
| url | http://journals.iium.edu.my/ejournal/index.php/iiumej/article/view/211 |
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