• Journal of Microbiology and Biotechnology
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• v.10 no.3
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• pp.293-299
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• 2000

Many methods have been developed for screening chemicals with hormonal activity. Using recombinant yeasts expressing either human estrogen receptor [Saccharomyces cerevisiae ER + LYS 8127 (YER)] or androgen receptor [S. cerevisiae AR + 8320 (YAR)], we evaluated the hormonal activities of several chemicals by induction of ${\beta}-galactosidase$ activity. The chemicals were $17{\beta}-estradiol$ (E2), testosterone (T), ${\rho}-nonylphenol$ (NP), bisphenol A (BPA), genistein (GEN), 2-bromopropane (2-BP), dibutyl phthalate (DBP), di-(2-ethylhexyl) phthalate (DEHP), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and butylparaben (BP). To assess the estrogenicity of NP, the result of the in vitro recombinant yeast assay was compared with an E-screen assay using MCF-7 human breast cancer cells and an uterotrophid assay using ovariectomized mice. In the YER yeast cells, E2, NP, BPA, GEN, and BP exhibited estrogenicity in a doseresponse manner, while TCDD did not. All the chemicals tested, except T, did not show androgenicity in the YAR yeast cell. The sensitivity of the yeast (YER) assay system to the estrogenic effect of NP was similar to that of the E-screen assay. NP was also estrogenic in the uterotrophic assay. However, in terms of convenience and costs, the yeast assay was superior to the E-screen assay or uterotrophic assay. These results suggest that the recombinant yeast assay can be used as a rapid tool for detecting chemicals with hormonal activities.

• Natural Product Sciences
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• v.9 no.4
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• pp.299-303
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• 2003

For the screening of inhibitors of sterol biosynthesis from natural products, a simple and rapid assay method was developed using recombinant yeast carrying human lanosterol synthase, main target of this assay method. Sterol biosynthesis inhibition activity was monitored only by the inhibition of growth of the recombinant yeast. By changing the substrate, this assay method can figure out which step is inhibited in the sterol biosynthesis by the test material. With this assay method total 102 plant samples were screened for their inhibitory activity of sterol biosynthesis. Among plant water extracts screened, 11 plant samples showed inhibitory activity on sterol biosynthesis in ergosterol (-) medium. For selection of the specific inhibitory materials, 11 plant samples were reassayed in ergosterol (+) medium. After all 5 plant samples, Abutilon avicennae Gaertn. (stem), Alnus japonica Steud. (stem), Amaranthus mangostanus L. (aerial part), Philadelphus schrenckii Pupr. (leaf) and Pimpinelia brachycarpa Nakai (aerial part), showed specific inhibitory activity.

• Korean Journal of Environmental Agriculture
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• v.27 no.4
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• pp.447-455
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• 2008

Recombinant yeast estrogenicity (YES) assay was used as a bioanalytical tool in order to screen $17{\beta}$-estradiol in the soil samples collected from different sites of South Korea. Solvent extraction and supercritical fluid extraction (SFE) methods were compared for the extraction of the estradiol from the soils. Most high detection of the estradiol based on YES assay was observed in the soils extracted with methanol. Different types of estrogenic hormones including $17{\beta}$-estradiol were suggested to be possibly exiting in the soils, since the methanol extracts of the soils showed an estrogenic activity that was not observed in the hexane extracts of the soil. SFE extracts showed estrogenic activity in some of the samples but methanol extract showed best activity.

• Journal of Food Hygiene and Safety
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• v.24 no.2
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• pp.124-127
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• 2009

The aim of this study is to evaluate the estrogenic activity of Cucurbita pepo seed extract which includes ${\beta}-sitosterol$ and other phytosterols. Sample was extracted from Cucurbita pepo seed by supercritical carbondioxide method and resuspended with ethanol. Estrogenic activity was measured by recombinant yeast assay which detects estrogenic activity using recombinant yeast with high level of estrogenic receptor. However, estrogenic activity of pumpkin seed extract was not found in this study. Based on this data, pumpkin seed extract will not cause estrogenic disturbance.

• Toxicological Research
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• v.22 no.1
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• pp.15-22
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• 2006

Many of endocrine disrupting chemicals induce effects via interaction with hormone receptors and responsive elements in target cells. We investigated endocrine disrupting effects of some food additives and contaminants including BHA, BHT, ethoxyquin, propionic acid, sorbic acid, benzoic acid, CPM, aflatoxin B1, cadmium chloride, genistein, TCDD and PCBs in yeast transformants expressing human steroid hormone receptors along with steroid responsive elements. The response limit of genetically recombinant yeast to $17{\beta}$-estradiol, testosterone and progesterone was $1{\times}10^{-16},\;1{\times}10^{-12}\;and\;1{\times}10^{-13}M$, respectively. BHT induced weak transcriptional activity in estrogen sensitive yeast, while BHA and sorbic acid interacted weakly with androgen receptor/responsive element. CPM induced transcriptional activities in all types of yeasts sensitive to steroid hormones. Zearalenone and genistein induced high transcriptional activation in estrogen sensitive yeast with relative potencies almost $10^8$ folds lower than $17{\beta}$-estradiol. TCDD induced transcriptional activation weakly in estrogen- and progesterone- sensitive yeasts. This study elucidated that recombinant yeast is a sensitive and high-throughput system and can be used for the direct assessment on chemical interactions with steroid receptors and responsive elements. Also, the present study raises the requirement of evaluation on the endocrine disrupting effects of BHT, BHA, sorbic acid, CPM and TCDD for their transcription activity in yeast screening system though weak in intensity.

• Journal of Veterinary Science
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• v.24 no.1
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• pp.15.1-15.13
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• 2023

Background: Inactivated vaccines are limited in preventing foot-and-mouth disease (FMD) due to safety problems. Recombinant virus-like particles (VLPs) are an excellent candidate for a novel vaccine for preventing FMD, given that VLPs have similar immunogenicity as natural viruses and are replication- and infection-incompetent. Objectives: The 3C protease and P1 polyprotein of type O FMD virus (FDMV) was expressed in yeast Hansenula polymorpha to generate self-resembling VLPs, and the potential of recombinant VLPs as an FMD vaccine was evaluated. Methods: BALB/c mice were immunized with recombinant purified VLPs using CpG oligodeoxynucleotide and aluminum hydroxide gel as an adjuvant. Cytokines and lymphocytes from serum and spleen were analyzed by enzyme-linked immunosorbent assay, enzyme-linked immunospot assay, and flow cytometry. Results: The VLPs of FMD were purified successfully from yeast protein with a diameter of approximately 25 nm. The immunization of mice showed that animals produced high levels of FMDV antibodies and a higher level of antibodies for a longer time. In addition, higher levels of interferon-γ and CD4⁺ T cells were observed in mice immunized with VLPs. Conclusions: The expression of VLPs of FMD in H. polymorpha provides a novel strategy for the generation of the FMDV vaccine.

• Biotechnology and Bioprocess Engineering:BBE
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• v.6 no.3
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• pp.173-178
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• 2001

Food yeast, Saccharomyces cerevisiae, is a safe organism with a long history of use for the production of biomass rich in high quality proteins and vitamins. AmA1, a seed storage albumin from Amaranthus hypochondriacus, has a well-balanced amino acid composition and high levels of essential amino acids and offers the possibility of further improving food animal feed additives. In order to find an effective means of expressing AmA1 in yeast, the gene was cloned into an episomal shuttle vector. Four different promoters were tested: the glyceraldehyde-3-phosphate dehydrogenase promoter, galactose dehydrogenase 10 promoter, alcohol dehydrogenase II promoter, and a hybrid ADH2-GPD promoter. The recombinant AmA1 genes were then introduced into the yeast Saccharomyces cerevisiae 2805. Northern and Western blot analyses of the yeast under appropriate conditions revealed that AmA1 was expressed by all four promoters at varying levels. An enzyme-linked immunosorbent assay demonstrated that the amount of AmA1 protein in the recombinant yeast was 1.3-4.3% of the total soluble proteins. The highest expression level was obtained from the hybrid ADH2-GPD promoter.

• BMB Reports
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• v.28 no.5
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• pp.437-442
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• 1995

The potency of yeast-derived methionyl-free human growth hormone (rhGH), which was obtained by removal of the N-terminal Met from methionyl-hGH, was estimated by in vitro and in vivo assays. In radio-receptor assay where the binding affinity of growth hormone to the receptor was estimated, the recombinant hGH showed 2.9 international units (IU) per mg of specific activity. In contrast, pitUitary-derived human growth hormone had a slightly lower receptor binding activity (2.5 IU/mg) compared with recombinant growth hormone. For the in vivo assay, efficacy of rhGH was tested by use of hypophysectomized rats, in which pituitary organs were surgically removed, resulting in the termination of growth hormone secretion. The weight-increase in rats by the injection of rhGH was almost identical to the result obtained by the injection of the same amount of pituitary-derived (international standard) hGH. A comparision of the secondary structures of rhGH and rMet-hGH by circular dichroism spectrophotometer demonstrated that the removal of the methionyl residue from rMet-hGH did not exert any effect on the structure of the growth hormone. In conclusion, methionyl-free human growth hormone produced from yeast was highly potent in biological activity and maintained a legitimate three dimensional structure.

• Biotechnology and Bioprocess Engineering:BBE
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• v.8 no.2
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• pp.162-165
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• 2003

In order to study the secretion of the human urokinase-type plasminogen activator, u-PA, from the yeast yarrowia lipolytica, three kinds of integrative expression vector were constructed. These vectors differed only in their secretion control legions, pre-, pre-dip-(dipeptide Stretch) or pre-dip-pro sequences of the alkaline extracellular protease, which were joined inflame to the human u-PA cDNA. The recombinant Y. lipolytica Strains, transformed with the expression vectors, secreted the hyperglycosylated u-PA. A fibrin plate assay of the culture supernatants showed that the hyperglycosylated u-PA proteins could catalyze fibrinolysis, and that the pre-dip sequence was the most efficient secretory signal for the secretion of the u-PA from Y. lipolyica. This result suggests that Y. lipolytica can be developed as a potential host for the production of recombinant human u-PA.

• Korean Journal of Microbiology
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• v.36 no.3
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• pp.236-241
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• 2000

A defensin is an inducible antibacterial peptide from a fleshfly and contains 40 residues basic peptide with six cysteines. For the consiruction of recombinant S cerevisiae expressing defensin, the structural gene coding for active defensin was chemically synthesized and fused in fiam to GAP promoter, MFul preprosequence and the GAL7 transcription terminator, generating a recombinant plasnlid pGMD18. S. ce~evisine 2805 Gells were transror~ned to uracil prototroph by the pGMDl8 arid the transformed cells showing antibacterial activity against 111. luteus TAM1056 were selected by growth inhibition zone assay. The optimal culture conditions for the unprovement of the defensin production of a selected tmdonnant were investigated. The optirmzed medium containing 0.4% yeast extract, 2% corn steep liquor, 2.5% glucose and 0.05% $C_2CO_3$, could be determined and the optimum lemperature. and initial pH could be detennnied as $28^{\circ}C$ and pH 3, ~mpectively. The optimized conditioiis revealed the trvofold Increase in the cell growth and the fourfold in the antibaclerial activity. coinpar-ed with tllc Yl'D medium.