Abstract
Background: DNA topoisomerase I (hTopoI) is an essential cellular enzyme that is found in all human cells. As this enzyme is upregulated in cancer cells exceedingly, it is used as a target for cancer chemotherapeutic drug development. As such, producing the in-house enzyme for the purpose to foster the search for more cost-effective and target specific hTopoI inhibitors is warranted. This study aims to compare the optimized conditions for the expression of hTopoI in KM71H (Mut S ) and X33 (Mut + ) strains of Pichia pastoris . P. pastoris transfected with an hTopoI recombinant vector was used for the optimization of a higher level of hTopoI expression. Results: In the process, fed-batch cultivation parameters that influence the expression of hTopoI, such as culture temperature, methanol induction and feeding strategy, were optimised in the transfected KM71H and X33 P. pastoris strains in a shake flask system. The cell density and total protein concentration (protein level) of transfected P. pastoris were compared to determine the optimum culture conditions for each transfected P. pastoris strain. A higher hTopoI level was observed in the transfected KM71H culture supernatant (2.26 ng/mL) when the culture was incubated in the optimum conditions. Conclusions: This study demonstrated that Mut S strain (KM71H) expressed and secreted a higher level of hTopoI heterologous protein in the presence of methanol compared to the Mut + strain; X33 (0.75 ng/mL). However, other aspects of optimization, such as pH, should also be considered in the future, to obtain the optimum expression level of hTopoI in P. pastoris .
Highlights
Human DNA topoisomerase I is an essential cellular enzyme in all living cells, including cancers [1]
The cell density at this incubation period was (11.48 ± 0.080) unit (p b 0.01), increased by approximately 56.6% compared to the cell density of same transformed yeast incubated at the same culture temperature for 12 h; (7.33 ± 0.060) unit
The total protein concentration of the transformed yeast increased by approximately 100.0% compared to the total protein concentration of the same transformed yeast cultivated at the same culture temperature for 12 h; (0.05 ± 0.009) μg/μL
Summary
Human DNA topoisomerase I (hTopoI) is an essential cellular enzyme in all living cells, including cancers [1]. The hTopoI regulates many events during the transcription cycle [8], whereby the cycle involves an intermediate covalent with the enzyme bound to the DNA complex (hTopoI-DNA-cc) This phenomenon allows the controlled rotation of the cleave DNA strand around the intact one. The stabilization of the intermediate covalent by drugs, oxidative lesions or other agents results in the formation of a single DNA strand break that can arrest transcription or replication when the enzyme collides with an RNA polymerase or a DNA polymerase, respectively. In the latter case, the single DNA strand break is converted into a double DNA strand break. Other aspects of optimization, such as pH, should be considered in the future, to obtain the optimum expression level of hTopoI in P. pastoris
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