Devi Vadiveloo, Sajetra
(2020)
Effects Of Camptothecin On Pichia
Pastoris Strain Smd1168h Expressing
Dna Topoisomerase I
On Agar Plates.
Project Report.
Pusat Pengajian Teknologi Industri.
(Submitted)
Abstract
DNA topoisomerase I (TopoI) is a ubiquitous enzyme, that is responsible for
releasing topological stress by introducing a temporary nick in one strand of the DNA
helix and later, resealing the single-stranded DNA. TopoI is involved in cell
proliferation; therefore, overexpression of this enzyme in a cell often mimics cancer
cells. Hence, this enzyme plays a major role in molecular biology studies for
developing various types of antineoplastic agents, such as camptothecin.
Camptothecin exhibits strong anti-inhibitory properties towards the catalytic activity
of TopoI by preventing the re-ligation of the nicked DNA, resulting in shear stress and
eventually, cell death. Pichia pastoris expression system is well-known for its ability
to produce human-like endogenous TopoI compared to other expression systems, e.g.
Escherichia coli, Saccharomyces cerevisiae and baculovirus. Currently, searching for
more effective compounds to reduce the toxicity of cancer treatments, while still
producing similar effects as current chemotherapy regimens is required. As such, this
research aims to investigate the inhibitory properties of camptothecin on the growth of
recombinant clones of multi-copy number insert of P. pastoris transformants
expressing human DNA topoisomerase I (SMD1168H-pPICZαA-hTopoI) grown on
yeast extract agar and microbiological agar plates, respectively. The agar plates
contained different concentrations of camptothecin (25 µM, 50 µM, 75 µM and 100
µM) and were left to incubate for 3 days. In conclusion, the highest inhibitory activity
of camptothecin was observed when both the recombinant clones were grown in
microbiological agar plate that contained 100 µM of camptothecin.
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