alexa Expression And Purification Of Human DNA Topoisomerase I In Pichia Yeast Expression System | 17290
ISSN: 2155-952X

Journal of Biotechnology & Biomaterials
Open Access

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5th World Congress on Biotechnology
June 25-27, 2014 Valencia Conference Centre, Valencia, Spain

Mooi Kwai Chan
Accepted Abstracts: J Biotechnol Biomater
DOI: 10.4172/2155-952X.S1.029
Abstract
Human DNA Topoisomerase I (Topo I) plays an important role in the transcription and replication processes. The enzyme can relax negative and positive supercoil DNA by introducing a transient nick on the single stranded DNA, rotation of the free stranded DNA and religation of the broken strand. As the enzyme has been used as the targets of anticancer drugs for chemotherapy, the enzyme can be developed as a molecular target for screening of potential anticancer compounds. Therefore, producing an in house Topo I through recombinant technology will facilitate our project in screening of anticancer compounds. In this study, the coding region of Top I from cDNA of MDA-MB 231 cell was amplified by PCR with Phusion enzyme. The cDNA which is 2298bp in length was ligated into a yeast expression vector, pPICZA-alpha and transformed into Top?10 electrocompetent cells. The positive clones with the desired insert were isolated and plasmid was purified for sequencing. The recombinant plasmid was linearized with Sac I restriction enzyme and transformed into pichia strain, GS 115 by electroporation. The positive integrated transformants was grown in BMGY(Buffered Glycerol-complex medium)and expression of the protein was induced in BMMY (Buffered Methanol-complex medium), pH 6.0 at 28 0 C, 250 rpm with addition of 0.5% (V/V) methanol every 24 hour. Activity of the recombinant enzyme was determined with DNA relaxation assay using 1 μl of the culture supernatant. The enzyme was purified with HiTrap CM FF weak cation exchanger column (GE healthcare, Sweden) using ?KTAprime plus protein purification system. SDS-PAGE and Western blot analyses showed the recombinant human Topo I with a molecular weight apparently of 85 kDa which is approximately 10 kDA less than the actual molecular weight of human Topo I. This might due to the proteolysis of the protein during the extracellular expression.
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