Expression And Purification Of Human DNA Topoisomerase I In Pichia Yeast Expression System | 17290
Journal of Biotechnology & Biomaterials
Like us on:
Our Group organises 3000+ Global Conferenceseries Events every year across USA, Europe & Asia with support from 1000 more scientific Societies and Publishes 700+ Open Access Journals which contains over 50000 eminent personalities, reputed scientists as editorial board members.
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
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
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.
Peer Reviewed Journals
Make the best use of Scientific Research and information from our 700 + peer reviewed, Open Access Journals