Multiple gene copy number increases total protein expression and enzyme activity of DNA topoisomerase I in Pichia pastoris
- Nur Adila Fadzil
- Shern Kwok Lim
- Ai Lan Chew
- Boon Yin Khoo
Affiliations: Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia (USM), 11800 Penang, Malaysia
- Published online on: August 9, 2022 https://doi.org/10.3892/wasj.2022.167
Copyright: © Fadzil
et al. This is an open access article distributed under the
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The present study aimed to employ an in vivo strategy for the establishment of multi‑copy gene constructs of human DNA topoisomerase I (hTopI) using the pPIC3.5K vector in Pichia. The clones with multi‑copy inserts (His+ transformants) that were able to survive in the highest concentration of Geneticin® were found to express the highest expression level of total protein and exhibited the highest target enzyme activity. The highest level of total protein found was 1.76 mg/ml in GS115‑pPIC3.5K‑hTop1, which was resistant to 1.00 mg/ml Geneticin at 48 h of incubation. The highest enzyme activity of hTopI was also observed in the culture expressed by GS115‑pPIC3.5K‑hTopI, which was resistant to 1.00 mg/ml Geneticin® (19.7x104 Ul/OD600). On the whole, the present study provides information regarding the production of target protein from recombinant Pichia using only a shaker flask system, which can be further developed as an in‑house resource for screening potential anticancer agents.