Molecular cloning of phytase gene from ASUIA279 and its expression in Pichia Pastoris system
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 li...
| 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/17046/ http://irep.iium.edu.my/17046/ http://irep.iium.edu.my/17046/4/219-1318-1-PB.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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