The Effect Of Gene Copy Number On The Expression And Activity Of Dna Topoisomerase I In Pichia Pastoris
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Date
2018-08
Authors
Nur Adila Fadzil
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Publisher
Universiti Sains Malaysia
Abstract
Human DNA topoisomerase I (hTopI) is an essential cellular enzyme in all living cells, whereby the enzyme is often upregulated in cancer cells. It is a target for chemotherapeutic drugs of the Camptothecin (CPT) family, which are mainly used in the systemic treatment of human cancers. This study was carried out to construct and clone the multi-copy number of hTopI expression cassette in Pichia pastoris, to investigate and compare the effects of multi-copy number of gene expression cassettes on the cell density, total cellular protein and hTopI activity in Pichia. Two strategies (in vitro and in vivo) were used to construct the multi-copy number of target gene in Pichia in this study. The construction of the multi-copy number of hTop1 expression cassette with pAO815 vector using in vitro strategy was not successful. Thus, the in vivo strategy was employed for the construction using pPIC3.5K vector. The multi-copy number of hTop1 gene in the pPIC3.5K vector was successfully transformed into the Pichia strain GS115 via in vivo strategy. The clones with multi-copy inserts (His+ transformants), with resistance to 0.25 mg/ml, 0.50 mg/ml, 0.75 mg/ml and 1.00 mg/ml Geneticin were also successfully selected from the agar plates. The cell density of the GS115-pPIC3.5K-hTopI was likely to be unaffected by the number of hTopI copies that were transformed into the host cells. However, the total protein expression and the enzyme activity produced by the recombinant clones were increased in accordance to the increased copy number of hTop1 in the host, whereby the clones that were able to survive on the highest concentration of Geneticin were found to express the highest level of total protein and showed the highest activity of target enzyme. The highest expression level of total protein was 1.76 mg/ml in GS115-pPIC3.5K-hTop1 which was resistant to 1.00 mg/ml Geneticin at 48 hours of incubation. The increment of gene copy number was also found to increase the activity of hTop1 produced in the host cells, whereby the highest enzyme activity of hTopI was observed in the culture expressed by GS115-pPIC3.5K-hTopI which was resistant to 1.00 mg/ml of Geneticinī (19.7 x 104 UL-1 OD600-1). Further study is warranted to investigate whether a sufficient amount of this protein can be produced from the recombinant Pichia using only shaker flask system that can be further developed as an in-house resource for potential drug screening.
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Keywords
Gene Copy