• Biotechnology and Bioprocess Engineering:BBE
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• 제6권3호
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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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• 제43권2호
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• pp.110-114
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• 2010

The yeast three-hybrid system (Y3H), a powerful method for identifying RNA-binding proteins, still suffers from many false positives, due mostly to RNA-independent interactions. In this study, we attempted to efficiently identify false positives by introducing a tetracycline operator (tetO) motif into the RPR1 promoter of an RNA hybrid expression vector. We successfully developed a tight tetracycline-regulatable RPR1 promoter variant containing a single tetO motif between the transcription start site and the A-box sequence of the RPR1 promoter. Expression from this tetracycline-regulatable RPR1 promoter in the presence of tetracycline-response transcription activator (tTA) was positively controlled by doxycycline (Dox), a derivative of tetracycline. This on-off control runs opposite to the general knowledge that Dox negatively regulates tTA. This positively controlled RPR1 promoter system can therefore efficiently eliminate RNA-independent false positives commonly observed in the Y3H system by directly monitoring RNA hybrid expression.

• Journal of Life Science
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• 제9권2호
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• pp.52-56
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• 1999

The Raf, a cytoplasmic serine/thereonine protein kinase, acts as an important mediator of signals involving cell proliferation, differentiation and development. Multiple regulatory elements should participate in the expression of D-raf, Drosophila homolog of human c-raf-1. In order to search regulatory factors involved in the D-raf promoter activation, we accomplished the yeast one-hybrid screening using D-raf promoter region from bp-330 to -309 with respect to the transcription initiation site as bait. After screening, sixteen independent positive clones of ${\beta}$-galactosidase activties were identified and sequenced. Two clones having 94-98% identity with daughterless and one clone having 93% identity with escargot by Blast search among these clones were screened.

• Journal of Microbiology and Biotechnology
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• 제13권4호
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• pp.537-543
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• 2003

The mouse interferon gene (MuIFN-${\gamma}$) was cloned and then used to transform Saccharomyces cerevisiae. Expressed MuIFN-$\{gamma}$ protein (MuIFN-${\gamma}$) was successfully secreted into culture medium due to the presence oi the signal peptide of rice amylase 1A. Two different promoters fused to MuIFN-${\gamma}$ were tested: glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter and a yeast hybrid ADH2-GPD (AG) promoter consisting of alcohol dehydrogenase II (ADH2) and GPD promoter. Using the hybrid promoter, the accumulation of MuIFN-${\gamma}$transcript was the highest after the 24 h cultivation, and then gradually decreased as the cultivation proceeded. However, both cell growth and recombinant MuIFN-${\gamma}$production reached their peaks after the 4-day cultivation. It was possible to produce 6.5 mg/l of MuIFN-${\gamma}$ without any changes in cell growth. Using GPD promoter, the MuIFN-${\gamma}$ transcript accumulation and the recombinant MuIFN-${\gamma}$ production followed the same pattern as the cell growth. However. compared to that of the hybrid promoter, the production of recombinant MuIFN-${\gamma}$ was 0.2 mg/l. The secreted MuIFN-${\gamma}$ had estimated molecular masses of 21 kDa and 23 kDa, which were larger than that of the encoded size due to glycosylation. The protection assay against the viral infection indicated that the recombinant MuIFN-${\gamma}$ was bioactive.

• 대한약학회:학술대회논문집
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• 대한약학회 2003년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2-2
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• pp.89.1-89.1
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• 2003

The signaling pathway of dopamine D$_2$ receptor was studied using yeast two-hybrid system. The 3rd cytoplasmic loop of rat D$_2$ receptor was fond to interact with ALY. The interaction in the yeast was observed only with the 3rd cytoplasmic loop of D$_2$ receptor but not with that of D$_3$ or D$_4$ dopamine receptor. The interaction between two proteins was also confirmed by GST pull-down assay. Co-expression of D$_2$ receptor and ALY enhanced the expression of Lef-1 promoter in C6 cells and the promoter of D$_2$ dopamine receptor itself.

• Applied Biological Chemistry
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• 제31권3호
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• pp.267-273
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• 1988

Transformed, hybrid strains of the yeast Saccharomyces capable of simultaneous secretion of both glucoamylase and ${\alpha}-amylase$ have been produced. These strains can carry out direct, one-step assimilation of starch with conversion efficiency greater than 93% during a 5 day growth period. One of the transformants converts 92.8% of available starch into reducing sugars in only 2 days. Glucoamylase secretion by these strains results from expression of one or more chromosomal STA genes derived from Saccharomyces diastaticus. The strains were transformed by a plasmid(pMS12) containing mouse salivary ${\alpha}-amylase$ cDNA in an expression vector containing yeast alcohol dehydrogenase promoter and a segment of yeast $2{\mu}$ plasmid. The major starch hydrolysis product produced by crude amylases found in culture broths is glucose, indicating that ${\alpha}-amylase$ and glucoamylase act cooperatively.

• Biotechnology and Bioprocess Engineering:BBE
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• 제9권4호
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• pp.292-296
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• 2004

The recombinant soluble human tumor necrosis factor-alpha (hTNF-$\alpha$) was expressed in a yeast Saccharomyces cerevisiae and its cytotoxicity was evaluated. A cDNA encoding hTNF-$\alpha$ was placed under the control of two different promoters: a glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter and a yeast hybrid ADH2-GPD promoter, consisting of alcohol dehydrogenase II (ADH2) and the GPD promoter. A Northern blot analysis revealed that, although variation in the expression level of hTNF-$\alpha$ existed among transformants, the higher expression was obtained with the GPD promoter. Expressed hTNF-$\alpha$ protein (rhTNF-$\alpha$) was successfully secreted into the culture medium, producing 2.5 mg per liter of culture filtrate, with no changes in cell growth. The bioassay for observing the cytotoxicity to the murine L929 fibroblast cell line, with serial dilution of rhTNF-$\alpha$, indicated that the secreted rhTNF-$\alpha$ was bioactive and its dose-response was improved eight to ten times over that of the E. coli-derived rhTNF-$\alpha$.

• 상하수도학회지
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• 제20권6호
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• pp.893-904
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• 2006

To improve sensitivity of biosensor as yeast two-hybrid detection system for estrogenic activity of suspected chemicals, we tested effects of several combinations of the bait and fish components in the two-hybrid system on Saccharomyces cerevisiae inducted a chromosome-integrated lacZ reporter gene that was under the control of CYC1 promoter and the upstream Gal4p-binding element $UAS_{GAL}$. The bait components that were fused with the Gal4p DNA binding domain are full-length human estrogen receptor ${\alpha}$ and its ligand-binding domain. The fish components that were fused with the Gal4p transcriptional activation domain were nuclear receptor-binding domains of co-activators SRC1 and TIF2. We found that the combination of the full-length human estrogen receptor ${\alpha}$ with the nuclear receptor-binding domain of co-activator SRC1 was most effective for the estrogen-dependent induction of reporter activity among the two-hybrid systems so far reported. The relative strength of transcriptional activation by representative natural and xenobiotic chemicals was well correlated with their estrogenic potency that had been reported with other assay systems.

• 대한약학회:학술대회논문집
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• 대한약학회 2003년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2-2
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• pp.89.2-89.2
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• 2003

Ral GDP dissociation stimulator (Ral GDS) has been found to be an effector protein of Ras, and Ral, a member of small GTP-binding protein (G protein) superfamily, has been suggested to act downstream of Ras. Ral GDP dissociation stimulator-like (RGL) shares 50% amino acid identity with Ral GDP dissociation stimulator, and assumed to possess similar functional role. Using yeast two-hybrid screening, we found that dopamine D3 receptor interacts with RGL. Since RGL is known to regulate the expression of c-fos promoter, effects of D3R on gene expression of c-fos promoter was studied. (omitted)

• BMB Reports
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• 제35권6호
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• pp.562-567
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• 2002

In order to find the cellular interaction factors of the Heliothis armigera nuclear polyhedrosis virus capsid protein VP39, a Heliothis armigera cell cDNA library was constructed. Then VP39 was used as bait. The host actin gene was isolated from the cDNA library with the yeast two-hybrid system. This demonstrated that VP39 could interact with its host actin in yeast. In order to corroborate this interaction in vivo, the vp39 gene was fused with the green fluorescent protein gene in plasmid pEGFP39. The fusion protein was expressed in the Hz-AM1 cells under the control of the Autographa californica multiple nucleopolyhedrovirus immediate early gene promoter. The host actin was labeled specifically by the red fluorescence substance, tetramethy rhodamine isothicyanete-phalloidin. Observation under a fluorescence microscopy showed that VP39, which was indicated by green fluorescence, began to appear in the cells 6 h after being transfected with pEGFP39. Red actin cables were also formed in the cytoplasm at the same time. Actin was aggregated in the nucleus 9 h after the transfection. The green and red fluorescence always appeared in the same location of the cells, which demonstrated that VP39 could combine with the host actin. Such a combination would result in the actin skeleton rearrangement.