Comparing the expression of human DNA topoisomerase I in KM71H and X33 strains of Pichia pastoris

Ang, Ruo Ping; Teoh, Leong Sin; Chan, Mooi Kwai; Miswan, Noorizan; Khoo, Boon Yin

doi:10.1016/j.ejbt.2016.01.007

Cited by 8 publications

(5 citation statements)

References 33 publications

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“…Next, the proper hosts were selected for porcine MG expression because the types of K. phaffii hosts can saliently affect the expression level of foreign genes. 21 The constructed pGAPZα A-MG plasmid was transformed into the K. phaffii hosts GS115, SMD1168, KM71, and X33, respectively, and the positive clones were selected in a YPD medium with 100 μg/mL of zeocin. The porcine MG secreted by different K. phaffii hosts in fermentation supernatants at the shaking-flask level was analyzed by SDS-PAGE (Figure 1A).…”

Section: ■ Results and Discussionmentioning

confidence: 99%

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Efficient Secretory Expression and Purification of Food-Grade Porcine Myoglobin in Komagataella phaffii

Zhang

Zhao

Wang

et al. 2021

J. Agric. Food Chem.

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Myoglobin (MG) is one of the eukaryotic heme-binding proteins that is closely associated with the real color and metallic taste of meat and can be used as a color additive in artificial meat alternatives. However, the traditional extraction methods are expensive and time-consuming and the heterologous biosynthesis of MG has never been reported. Herein, we achieved the secretory expression of porcine MG by engineered Komagataella phaffii using the suitable host (X33), signal peptide (α-factor signal peptide), and modified constitutive promoter (G1 promoter). In addition, the fermentation conditions for MG production were optimized at shaking-flask level (BMGY medium with 40 mg/L of hemin, 30 °C) and at fermenter level (30% DO, feeding 150 mg/L of hemin), resulting in the highest titer of 285.42 mg/L MG in fed-batch fermentations. Furthermore, a purification method for food-grade MG was developed, which can obtain 0.22 mol of heme/mol of MG with 88.0% purity and 66.1% recovery rate.

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Section: ■ Results and Discussionmentioning

confidence: 99%

“…Next, the proper hosts were selected for porcine MG expression because the types of K. phaffii hosts can saliently affect the expression level of foreign genes . The constructed pGAPZα A-MG plasmid was transformed into the K.…”

Section: Resultsmentioning

confidence: 99%

Efficient Secretory Expression and Purification of Food-Grade Porcine Myoglobin in Komagataella phaffii

Zhang

Zhao

Wang

et al. 2021

J. Agric. Food Chem.

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“…Finally, destabilases exert antibacterial activity by enzymatically cleaving a glycosidic linkage within the peptidoglycan of bacterial cell walls [68] . Further optimisation of experimental conditions, such as culture temperature, methanol concentration, and vector feeding strategy, is required to achieve expression of Tc his, T scp, and T des [69] .…”

Section: Discussionmentioning

confidence: 99%

Structural and functional analyses of nematode-derived antimicrobial peptides support the occurrence of direct mechanisms of worm-microbiota interactions

Rooney,

Rivera-de-Torre,

et al. 2024

Computational and Structural Biotechnology Journal

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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).…”

Section: Discussionmentioning

confidence: 99%

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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Comparing the expression of human DNA topoisomerase I in KM71H and X33 strains of Pichia pastoris

Cited by 8 publications

References 33 publications

Efficient Secretory Expression and Purification of Food-Grade Porcine Myoglobin in Komagataella phaffii

Efficient Secretory Expression and Purification of Food-Grade Porcine Myoglobin in Komagataella phaffii

Structural and functional analyses of nematode-derived antimicrobial peptides support the occurrence of direct mechanisms of worm-microbiota interactions

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

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