• Journal of Microbiology and Biotechnology
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• v.8 no.1
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• pp.42-48
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• 1998

In order to maximize the secretory expression of human serum albumin (HSA) in the yeast Saccharomyces cerevisiae, a series of HSA expression vectors were constructed with a combination of different promoters, 5' untranslated regions (5'UTR), and secretion signal sequences. The expression vector composed of the galactose-inducible promoter GALl0, the natural 5'UTR, and the natural signal sequence of HSA directed the most efficient expression and secretion of HSA among the constructed vectors when introduced into several S. cerevisiae strains. Although the major form of HSA expressed and secreted in the yeast transformants was the mature form of 66 kDa, the truncated form of 45 kDa was also detected both in the cell extract and in the culture supernatant. The level of the intact HSA protein in the culture supernatant reached up to 30 mg/l at 24 h of cultivation in a shake-flask culture but began to decrease afterwards, indicating that the secreted HSA protein was unstable in a prolonged culture of yeast.

• Korean Journal of Microbiology
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• v.42 no.3
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• pp.195-198
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• 2006

Lethal toxin is a critical virulence factor of anthrax. It is composed two protein: protective antigen (PA) and lethal factor (LF). PA binds to specific cell surface receptors and, forms a membrane channel that mediates entry of LF into the cell. LF is a zinc-dependent metalloprotease, which cleaves MKKs [MAPK (mitogen-activated protein kinase) kinases] at peptide bonds very close to their N-termini. In this study, we suggest application of cell-based assays in the early phase of drug discovery, with a particular focus on the use of yeast cells. We constructed MEK1 expression system in yeast to determine LF activity and approached cell-based assay system to screen inhibitors, in which the results covering the construction of LF-substrate in yeast expression vector, expression, and LF-mediated proteolysis of substrate were described. These results could provided the basic steps in design of cell-based assay system with the high efficiency, rapidly and easy way to screening of inhibitors.

• Korean Journal of Microbiology
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• v.32 no.4
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• pp.271-279
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• 1994

To develop a yeast strain which stably secretes both $\alpha$-amylase and glucoamylase and therefore is able to convert starch directly to ethanol, a mouse salivary $\alpha$-amylase cDNA gene with a yeast alcohol dehydrogenase I promoter has been introduced into the cell of a Saccharomyces diactaticus hybrid strain secreting only glucoamylase. To secrete both enzymes more stably without loss of the $\alpha$-amylase gene during a cell-multiplication, an integrating plasmid vector containing $\alpha$-amylase gene was constructed and introduced into the yeast cell. The results showed that the linearized form of the integrating vector was superior in the transformation efficiency and the rate of the expression of the $\alpha$-amylase gene than the circular type of the vector. The yeast transformant having a linearized plasmid vector exhibited higher mitotic stability than the yeast transformant habouring episomat plasmid vector. The transformant containing the linearized vector producing both $\alpha$-amylase and glucoamylase exhibited 2-3 times more amylolytic activity than the original untransformed strain secreting only glucoamylase.

• Microbiology and Biotechnology Letters
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• v.21 no.3
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• pp.214-220
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• 1993

The mating type a of yeast, Saccharomyces cerevisiae mutant with hmla2-102 and sir3-8ts was changed to type alpha by changing the growth temperature from 25C to 35C. A temperature-sensitive expression vector system was constructed using mating factor alpha1 (Mfalpha1) gene encoding alpha factor which is expressed in the type alpha cells. Vectors with different copy numbers were constructed by joining the promoter and pre or prepro-secretion single sequence of Mfalpha1 to promoterless PHO5' gene as a reporter gene.

• 한국생물공학회:학술대회논문집
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• 2000.04a
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• pp.153-156
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• 2000

Xylan, the major hemicellulose component of many plants, occurs naturally in a partially acetylated form and lignin, the most resistant component in plant cell wall degradation, is also attached to ${\beta}-1,4-linked-D-xylose$ backbone through the ester linkage. Esterases are required to release the esterified substituent and acetyl esterases are important in the complete degradation of acetylated polysaccharides, like pectins and xylans. The gene(Axe) encoding acetyl xylan estarase(AXE) was isolated from genomic ${\lambda}$ library from Aspergillus ficuum. Nucleotide sequencing of the Axe gene indicated that the gene was separated with two intervening sequences and the amino acid sequence comparison revealed that it was closely related to that from A. awamori with the 92 % indentity. Heterologous expression of AXE was conducted by using YEp352 and Saccharomyces cerevisae 2805 as a vector and host expression system, respectively. The Axe gene was placed between GAL1 promoter and GAL7 terminator and then this recombinant vector was used to transform S. cerevisiae 2805 strain. Culture filtrate of the transformed yeast was assayed for the presence of AXE activity by spectrophotometry and, comparing with the host strain, four to five times of enzyme activity was detected in culture filtrate of transformed yeast.

• Journal of Microbiology and Biotechnology
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• v.22 no.11
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• pp.1557-1567
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• 2012

Glutathione reductase (GR, E.C. 1.6.4.2) is an important enzyme that reduces glutathione disulfide (GSSG) to a sulfydryl form (GSH) in the presence of an NADPH-dependent system. This is a critical antioxidant mechanism. Owing to the significance of GR, this enzyme has been examined in a number of animals, plants, and microbes. We performed a study to evaluate the molecular properties of GR (OsGR) from rice (Oryza sativa). To determine whether heterologous expression of OsGR can reduce the deleterious effects of unfavorable abiotic conditions, we constructed a transgenic Saccharomyces cerevisiae strain expressing the GR gene cloned into the yeast expression vector p426GPD. OsGR expression was confirmed by a semiquantitative reverse transcriptase polymerase chain reaction (semiquantitative RT-PCR) assay, Western-blotting, and a test for enzyme activity. OsGR expression increased the ability of the yeast cells to adapt and recover from $H_2O_2$-induced oxidative stress and various stimuli including heat shock and exposure to menadione, heavy metals (iron, zinc, copper, and cadmium), sodium dodecyl sulfate (SDS), ethanol, and sulfuric acid. However, augmented OsGR expression did not affect the yeast fermentation capacity owing to reduction of OsGR by multiple factors produced during the fermentation process. These results suggest that ectopic OsGR expression conferred acquired tolerance by improving cellular homeostasis and resistance against different stresses in the genetically modified yeast strain, but did not affect fermentation ability.

• 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.

• Microbiology and Biotechnology Letters
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• v.14 no.2
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• pp.155-160
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• 1986

$\alpha$-Amylase gene of B. amyloliquefaciens was cloned to E. coli-yeast shuttle vector YEp-13 and expressed in E. coli. Chromosomal DNA of B. amyloliquefaciens was partially digested with Sau3Al and YEp13 plasmid was cleaved with BamH1. The hybrid plasmid, pHA28, was constructed by shotgun method and transformed to E. coli C600 and HB101. The amount of $\alpha$-amylase produced by transformants of E. coli was about 20% to 30% of that produced by B. amyloli-quefaciens. About 65% of $\alpha$-amylase produced by transformant was secreted into periplasm and the others were located in cytoplasm. $\alpha$-Amylase production was maximal when transformants were cultivated for 15hr to 20hr. As the result of agarose gel electrophoresis, pHA28 plasmid was found to be various in its size. This result suggested that pHA28 plasmid was segregated.

• Journal of Microbiology and Biotechnology
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• v.1 no.4
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• pp.266-273
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• 1991

Hirudin, a 65-amino acid protein isolated from the salivary gland of the bloodsucking leech, Hirudo medicinalis, is a potent thrombin-specific inhibitor and blocks the thrombin-mediated conversion of fibrinogen to fibrin in clot formation. We have studied the gene expression and secretion of hirudin in yeast. Saccharomyces cerevisiae. A gene coding for hirudin was synthesized based on the amino acid sequence and cloned into a yeast expression vector $YEG{\alpha}-1$ containing the ${\alpha}-mating$ factor pre-pro leader sequence and galactose-inducible promoter, GALl0. Recombinant S. cerevisiae was found to secrete biologically active hirudin into the extracellular medium. The secreted recombinant hirudin was recovered from the culture medium and purified with ultrafiltration and reverse phase high performance liquid chromatography. Approximately 1 mg of hirudin per liter was produced under suboptimal culture conditions and brought to about 90% purity in two steps of purification.

• Microbiology and Biotechnology Letters
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• v.23 no.2
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• pp.138-144
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• 1995

The cDNA, encoding human lysozyme (HLY) which was isolated from a human placenta cDNA library, has been well characterized (Yoshimura et al., 1988). Based on the communication, we have prepared an artificial HLY gene from chemically synthesized 38-oligomer with high codon usage in Saccharomyces cerevisiae. For directing the synthesis and secretion of HLY in S. cerevisiae, an expression vector, pHKl was constructed by inserting the HLY gene, containing a synthetic HLY secretion signal sequence, between the yeast GAP promoter and PH05 terminator. From a lysoplate assay, we have confirmed an yeast transformant harboring a pHK1 which makes a clearing zone on the overlayed Micrococcus luteus. This result means a chemically synthesized HLY gene which was normally expressed and secreted in yeast.