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
2011
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| Subjects: | |
| Online Access: | http://irep.iium.edu.my/17110/ http://irep.iium.edu.my/17110/ http://irep.iium.edu.my/17110/1/molecular_cloning.pdf |
| Summary: | 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. |
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