Molecular cloning and production of recombinant phytase from Bacillus Subtilis ASUIA243 in Pichia Pastoris

• Main Authors: Mohd Dali, Nor Soleha, Nuge, Tamrin, Mahamad Maifiah, Mohd Hafidz, Yusof, Faridah, Meor Hussin, Anis Shobirin, Farouk, Abd Alaziem, Mohd. Salleh, Hamzah
• Format: Article
• Language: English
• Published: IIUM Press 2011
• Subjects: QH426 Genetics
• Online Access: http://irep.iium.edu.my/17110/1/molecular_cloning.pdf; http://irep.iium.edu.my/17110/; http://www.iium.edu.my/ejournal/index.php/iiumej/article/view/211/259
• Institution: Universiti Islam Antarabangsa 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.