Chan, Mooi Kwai
(2017)
Expression Of Recombinant Human Dna Topoisomerase In Pichia Pastoris And Analysis Of Its Activity.
PhD thesis, Universiti Sains Malaysia.
Abstract
This study was aimed to establish a method to produce in-house recombinant human DNA topoisomerase. The DNA topoisomerase I (Topo I) and DNA topoisomerase II (Topo II) coding regions were amplified from the cDNA of breast cancer cells, MDA-MB-231. Topo I and Topo II coding region were ligated into expression vector, pPICZ--A and pPICZ--C, respectively. Subsequently, the constructed recombinant vectors were transformed into four types of Pichia pastoris strain, X-33, GS115, SMD1168H and KM71H. PCR screening for recombinant clones was performed and followed by selection of multicopy clones using agar plates containing increasing concentrations of Zeocin. The selected multicopy clones were then induced for enzymes expression in a shake flask system. Topo I was expressed relatively higher in GS115 than other Pichia strains.
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