Background: Human 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 speed up the search for more cost-effective and target
specific hTopoI inhibitors is warranted. This study aims to compare the optimised 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.
