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KMID : 0364820200560010044
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Korean Journal of Microbiology
2020 Volume.56 No. 1 p.44 ~ p.53
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Prospective application of Pichia pastoris-expressed DNA topoisomerase I in DNA relaxation assay for growth inhibitor screening
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Kwai Chan Mooi
Xuan Wai Wen
Ping Ang Ruo
Lan Chew Ai
Yin Khoo Boon
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Abstract
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The expression of in-house DNA topoisomerase I (Topo1) was optimized in order to develop a gel-based DNA relaxation assay for the screening of potential bioactive compounds.
Results showed that nearly 4-fold increment in total protein level was achieved in the culture supernatant with 1.0% (v/v) methanol feeding every 24 h and cultivated for 72 h at 28°C.
Crude recombinant TopoI was shown to relax the supercoiled plasmid DNA completely in the developed gel-based assay and reactions were not affected by DMSO addition and the features on electrophoresed gel could be improved by chloroform: isoamylalcohol extraction. The recombinant protein was then purified and the enzymatic activity of the recombinant TopoI in the crude and purified culture supernatant was studied and compared with a commercial TopoI. DNA relaxation assay showed that the purified recombinant TopoI is a more reliable source with an activity of approximately 20~30 U/μl. The developed assay was finally evaluated by determining the inhibitory effects of flavonoids and positive control Camptothecin on the recombinant TopoI catalytic activity. The efficiency of the gel-based assay using purified recombinant TopoI was further evaluated against a cell-based cytotoxicity assay in which the inhibitory effects on cell growth by the flavonoids and Camptothecin were assessed. The similar inhibitory trends shown by both assays demonstrated that potential inhibitors can be preliminarily screened using a gel-based assay prior to identification of the inhibitors using a cell-based assay to detect the DNA intercalating agents that interrupt the TopoI catalytic activity.
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KEYWORD
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Pichia pastoris, catalytic activity inhibition, cytotoxicity assay, DNA relaxation assay, DNA topoisomerase I, growth inhibition
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