Comparing the expression of human DNA topoisomerase I in KM71H and X33 strains of Pichia pastoris
Human is an essential cellular enzymethat 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-effecti...
Saved in:
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Published: |
Elsevier
2016
|
| Subjects: | |
| Online Access: | http://eprints.usm.my/36835/ http://dx.doi.org/10.1016/j.ejbt.2016.01.007 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| Institution: | Universiti Sains Malaysia |
| Summary: | Human is an essential cellular enzymethat 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 (MutS) 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 thatMutS 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. |
|---|