Expression of stable and active human DNA topoisomerase I in Pichia pastoris

Chan, Mooi Kwai; Lim, Shern Kwok; Miswan, Noorizan; Chew, Ai Lan; Noordin, Rahmah; Khoo, Boon Yin

doi:10.1016/j.pep.2017.09.003

Cited by 16 publications

(14 citation statements)

References 14 publications

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“…Cheng et al introduced a targeting PEG-folic acid (PEG-FA) on the surface of PDA, and demonstrated that PEG-FA modified NPs exhibited higher antitumor efficacy against cancer cells with high-expression of folic acid receptors in vivo due to FA-mediated targeting [ 32 ]. Also, cyclic peptides containing RGD (Arg-Gly-Asp) motif can be modified on the surface of PDA through a PEG chain [ 33 ], which can be used to target cells overexpressing α v β 3 [ 34 ]. Taken together, the encapsulation of ICG in LAP nanodisks and then the coating of PDA on the surface may improve the stability of ICG, provide additional photothermal conversion efficacy, and facilitate the targeting modification of a nanoplatform.…”

Section: Introductionmentioning

confidence: 99%

Loading of Indocyanine Green within Polydopamine-Coated Laponite Nanodisks for Targeted Cancer Photothermal and Photodynamic Therapy

Liu

et al. 2018

Nanomaterials

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The combination of photothermal therapy (PTT) and photodynamic therapy (PDT) in cancer treatment has attracted much attention in recent years. However, developing highly efficient and targeted therapeutic nanoagents for amplifying PTT and PDT treatments remains challenging. In this work, we developed a novel photothermal and photodynamic therapeutic nanoplatform for treatment of cancer cells overexpressing integrin αvβ3 through the coating of polydopamine (PDA) on indocyanine green (ICG)-loaded laponite (LAP) and then further conjugating polyethylene glycol-arginine-glycine-aspartic acid (PEG-RGD) as targeted agents on the surface. The ICG/LAP–PDA–PEG–RGD (ILPR) nanoparticles (NPs) formed could load ICG with a high encapsulation efficiency of 94.1%, improve the photostability of loaded ICG dramatically via the protection of PDA and LAP, and display excellent colloidal stability and biocompatibility due to the PEGylation. Under near-infrared (NIR) laser irradiation, the ILPR NPs could exert enhanced photothermal conversion reproducibly and generate reactive oxygen species (ROS) efficiently. More importantly, in vitro experiments proved that ILPR NPs could specifically target cancer cells overexpressing integrin αvβ3, enhance cellular uptake due to RGD-mediated targeting, and exert improved photothermal and photodynamic killing efficiency against targeted cells under NIR laser irradiation. Therefore, ILPR may be used as effective therapeutic nanoagents with enhanced photothermal conversion performance and ROS generating ability for targeted PTT and PDT treatment of cancer cells with integrin αvβ3 overexpressed.

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Section: Introductionmentioning

confidence: 99%

Loading of Indocyanine Green within Polydopamine-Coated Laponite Nanodisks for Targeted Cancer Photothermal and Photodynamic Therapy

Liu

et al. 2018

Nanomaterials

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“…The yeast-based strategy described in the present study was developed when constructing Pichia with multiple copy numbers of TOPOI for the expression and purification of the target enzyme (13,20). GS115 and SMD1168 yeasts were found to be better than Pichia strains in accommodating the exogenous recombinant TOPOI expression, and an enzyme activity of ~3.02×10 5 U/l of crude culture was obtained in the recombinant yeast; however, only SMD1168 was able to stably express the enzyme in the culture supernatant at room temperature (13). This prototype development is based on our present original research, and provides innovation and novelty to screening processes within the medical biotechnology sector.…”

Section: Discussionmentioning

confidence: 99%

“…A Pichia pastoris strain clone of SMD1168H carrying TOPOI in a pPICZαA plasmid was generated in our previous study (13), and was referred to as SMD1168H-TOPOI. This clone was used for the development of the yeast-based screening assay in the present study.…”

Section: Methodsmentioning

confidence: 99%

“…Yeast culture stocks (SMD1168H, SMD1168H-pPICZαA and SMD1168H-TOPOI), which were constructed and stored in glycerol at −80°C as previously described (13), were retrieved and enriched using 5 ml buffered glycerol-complex medium (BMGY) in a universal bottle. The transformed yeast strain was incubated overnight at 15–20°C in an incubator shaker at 250 rpm.…”

Section: Methodsmentioning

confidence: 99%

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Potential use of Pichia pastoris strain SMD1168H expressing DNA topoisomerase I in the screening of potential anti‑breast cancer agents

et al. 2019

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Cancer chemotherapy possesses high toxicity, particularly when a higher concentration of drugs is administered to patients. Therefore, searching for more effective compounds to reduce the toxicity of treatments, while still producing similar effects as current chemotherapy regimens, is required. Currently, the search for potential anticancer agents involves a random, inaccurate process with strategic deficits and a lack of specific targets. For this reason, the initial in vitro high-throughput steps in the screening process should be reviewed for rapid identification of the compounds that may serve as anticancer agents. The present study aimed to investigate the potential use of the Pichia pastoris strain SMD1168H expressing DNA topoisomerase I (SMD1168H-TOPOI) in a yeast-based assay for screening potential anticancer agents. The cell density that indicated the growth of the recombinant yeast without treatment was first measured by spectrophotometry. Subsequently, the effects of glutamate (agonist) and camptothecin (antagonist) on the recombinant yeast cell density were investigated using the same approach, and finally, the effect of camptothecin on various cell lines was determined and compared with its effect on recombinant yeast. The current study demonstrated that growth was enhanced in SMD1168H-TOPOI as compared with that in SMD1168H. Glutamate also enhanced the growth of the SMD1168H; however, the growth effect was not enhanced in SMD1168H-TOPOI treated with glutamate. By contrast, camptothecin caused only lower cell density and growth throughout the treatment of SMD1168H-TOPOI. The findings of the current study indicated that SMD1168H-TOPOI has similar characteristics to MDA-MB-231 cells; therefore, it can be used in a yeast-based assay to screen for more effective compounds that may inhibit the growth of highly metastatic breast cancer cells.

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“…Each copy of hTopI was carried by pPIC3.5K plasmid (9.0 kb; Invitrogen, Carlsbad, USA). In the process, the complete nucleotide sequence encoded hTopI that had been generated in a previous study [19] was excised from a pPICZα-A-hTopI plasmid with the restriction endonucleases EcoRI (Thermo Fisher Scienti c, Waltham, USA) and NotI (Thermo Fisher Scienti c, Waltham, USA) in two separate enzymatic reactions. The sequence was then inserted into the pPIC3.5K plasmid at the EcoRI and NotI sites.…”

Section: Production Of a Multi-copy Number Insert In Pichia By An In mentioning

confidence: 99%

Quercetin and a plant substance, identified using a multi-copy number of DNA topoisomerase I in recombinant Pichia pastoris, inhibited MDA-MB-231 proliferation via different manners.

Zhao

Pei

Fadzil

et al. 2021

Preprint

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The study aimed to employ an in vivo strategy to construct a multi-copy number of human DNA topoisomerase I (hTopI) gene using pPIC3.5K vector in GS115 strain of Pichia. The yeast transformant (GS115-pPIC3.5K-hTopI) was then used to investigate the preliminary growth effect of a pure compound (quercetin) and a standardised subfraction of ethanolic red onion peel extract (F1). The underlying mechanisms of quercetin and F1 were then tested on MDA-MB-231 for the cell cycle profile and apoptosis by flow cytometry, and the mRNA expression of CYP genes by real-time PCR. His + yeast transformants (clones) with multi-copy inserts resistant to various concentrations of Geneticin were successfully selected in the study. The clones’ cell density was likely to be unaffected; only the total protein expression and enzyme activity were increased following the increased copy number of hTop1 in the host. The clone that showed the target enzyme's highest activity is said to respond specifically to growth inhibitors, whereby both quercetin and F1 were proven to be potential growth inhibitors as assessed by the MTT assay. In the process, quercetin reduced cell proliferation by inducing apoptosis and cell cycle arrest (S phase only), whereas F1 reduced cell proliferation by inducing cell cycle arrest only (S and G2 phases). Quercetin and F1 induced CYP1A1 and CYP1B1 (carcinogenicity) gene mRNA expression, but only F1 induced CYP2S1 (cytotoxicity) gene mRNA expression in the treated cells, suggesting that both quercetin and F1 inhibited the cell proliferation of MDA-MB-231 via different manners. The newly developed GS115-pPIC3.5K-hTopI can be used to select various potential substances for breast cancer treatment in the future.

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Expression of stable and active human DNA topoisomerase I in Pichia pastoris

Cited by 16 publications

References 14 publications

Loading of Indocyanine Green within Polydopamine-Coated Laponite Nanodisks for Targeted Cancer Photothermal and Photodynamic Therapy

Loading of Indocyanine Green within Polydopamine-Coated Laponite Nanodisks for Targeted Cancer Photothermal and Photodynamic Therapy

Potential use of Pichia pastoris strain SMD1168H expressing DNA topoisomerase I in the screening of potential anti‑breast cancer agents

Quercetin and a plant substance, identified using a multi-copy number of DNA topoisomerase I in recombinant Pichia pastoris, inhibited MDA-MB-231 proliferation via different manners.

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