• KSBB Journal
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• v.11 no.5
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• pp.543-549
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• 1996

Recombinant Saccharomyces cerevisiae BZ-pJ containing the gene coding for metallothionein, a metalbinding protein was grown in the medium with high cadmium concentrations to study the characteristics of growth and cadmium uptake. High concentrations of cadmium reduced cell growth and final cell density and increased the lag phase periods of the recombinant yeast. Addition of 10 mg $Cd^{2+}$/L to the growth medium remarkably decreased a lag period and enhanced the specific cadmium uptake to 52.6 mg $Cd^{2+}$/g dry cell. The effect of copper addition was further investigated in the medium of 680 mg Cd2+/L. An increase in copper concentration from 11.0 to 33.3 mg/L enhanced the specific cadmium uptake from 17.0 to 42.0 mg Cd2+/g dry cell.

• Biomolecules & Therapeutics
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• v.4 no.4
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• pp.297-300
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• 1996

LBD-001, a recombinant human interferon $\gamma$ produced by genetically engineered yeast as a host system, was administered intraperitoneally to Sprague-Dawley male rats from premating to mating period at least for 60 days and to female rats from at least for 2 weeks before mating to early gestation period (from day 0 to 7 of gestation) at dose levels of $0.35\times10^6, 0.39\times10^6, and 1.38\times10^6$ I.U./kg/day. In the positive control group, ethynylestradiol ($EE_2$; 40 $\mu\textrm{g}$/kg/day) was subcutaneously administered only to female rats during the early gestation period. Effects of the test agents on reproductive performances of the male or female rats and embryonic development were as followings; (1) No significant changes by the treatment of LBD-001 were observed in general behaviors, body weight, food and water consumption, and necropsy of parent animals. However, significant decreases of body weight, food consumption, and water consumption were observed in ($EE_2$ -treated female rats. (2) Mating performances and fertility of parent animals were not significantly affected by the treatment of LBD-001. In ($EE_2$ -treated females, however, the fertility was completely inhibited. (3) No changes in resorption rate and external abnormality of F1 fetuses were observed by the treatment of LBD-001. The results show that LBD-001 at the dose of $1.38\times10^6$ I.U./kg/day or less does not affect general toxicity and reproductive function of parent animals and embryonic development of F1 fetuses.

• Biomolecules & Therapeutics
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• v.4 no.4
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• pp.301-309
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• 1996

LBD-001, a recombinant human interferon ${\gamma}$ produced by genetically engineered yeast as a host system, was intravenously administered to pregnant female rats (Sprague-Dawley) from day 7 to 17 of gestation at dose levels of 0.35$\times$10$^{6}$ , 0.69$\times$10$^{6}$ , and 1.38$\times$10$^{6}$ I.U./kg/day. As the control groups, hydrocortisone sodium succinate (5 or 10 mg/kg/day) was also similarly administered. Teratological effects of the test agents on fetuses and development of offsprings (F1 rats) were investigated. (1) No significant changes by the treatment of LBD-001 were observed in body weight, food and water consumption, feeding and nursing behaviors, and autopsy of pregnant or lactating mother rats. However, in hydrocortisone sodium succinate (10 mg/kg/day)-treated group, significant decreases of body weight on day 16, 18, and 20 of gestation and food consumption on day 20 of gestation and outstanding atrophy of thymus and adrenals were observed in two rats autopsied on day 20 of gestation. (2) No significant changes in resorption rate, skeletal or visceral development of fetuses, and physical or sensory development of offsprings (Fl) by the treatment of LBD-001 were detected. In hydrocortisone sodium succinate (10 mg/kg/day)-treated group, however, there were significant decreases of body weight of fetuses, delay of ossification, temporary delay of body weights of offsprings (F1) on day 1 and 3 of lactation, and increased tendency of stillborn rate and malformation rate of bone. The results show that LBD-001 at the dose of 1.38$\times$10$^{6}$ I.U./kg/day or less is not teratogenic in organogenesis of fetuses and the development of offsprings (F1). Meanwhile, hydrocortisone sodium succinate (10 mg/kg/day) seems to delay ossification of fetuses and temporarily retard the development of offsprings (Fl).

• Journal of Life Science
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• v.18 no.2
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• pp.287-290
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• 2008

In order to obtain yeast cells producing a bacteriocin, Subpeptin JM4-A or Subpeptin JM4-B, the 48 bp oligonucleotides corresponding to Subpeptin JM4-A and Subpeptin JM4-B genes including codon for start and stop were chemically synthesized and cloned into pAUR123, an yeast expression vector. Transformed yeast cells exhibited growth inhibition of Bacillus subtilis, Escherichia coli and Pseudonomas aeruginosa. This result indicates that yeast cells producing Subpeptin JM4-A or Subpeptin JM4-B possess bacteriocidal properties against both Gram positive B. subtilis and Gram negative E. coli and P. aeruginosa cells. The recombinant yeast strains constructed in this study can be applied in the food preservative or. animal foodfeed.

• Microbiology and Biotechnology Letters
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• v.36 no.3
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• pp.221-226
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• 2008

To produce human ferritins in yeast, human H-chain and L-chain ferritins were amplified from previously cloned vectors. Each amplified ferritin gene was inserted into the pYES2.1/V5-His-TOPO yeast expression vector under the control of the GAL1promoter. Western blot analysis of the recombinant yeast cells revealed that H-and L-chain subunits of human ferritin were expressed in Saccharomyces cerevisiae. Atomic absorption spectrometry (AAS) analysis demonstrated that the intracellular content of iron in the ferritin transformant was 1.6 to 1.8-fold higher than that of the control strain. Ferritin transformants could potentially supply iron-fortified nutrients for food and feed.

• 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.

• Biotechnology and Bioprocess Engineering:BBE
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• v.9 no.4
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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-$\alpha$.

• Microbiology and Biotechnology Letters
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• v.32 no.1
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• pp.60-66
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• 2004

The cycloinulooligosaccharide fructanotransferase(CFTase) gene (cft) from Paenibacillus polymyxa was subcloned into the E. coli-yeast shuttle vector, pYES2.0 (GALI promoter). The constructed plasmid, pYGCFT (9.9 kb) was introduced into S. cerevisiae SEY2102 cell and then the yeast transformant was selected on the synthetic defined media lacking uracil Based on the cyclofructan(CF) spots on thin-layer chromatogram, the gene under the control of GALI promoter was successfully expressed in the yeast transformant. The recombinant CFTase was not secreted into the medium and was predominantly localized in the periplasmic space. CF was started to be produced after 3h of enzymatic reaction with inulin. The pH and temperature optimum for the CF production from inulin was pH 8.0 and 45$^{\circ}C$, respectively. Enzyme activity was stably maintained up to the pH of 10.0. The examination of the inulin sources revealed that a dahlia tuber and Jerusalem artichoke were the best for the production of CF.

• BMB Reports
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• v.29 no.4
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• pp.386-392
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• 1996

A Saccharomyces cerevisiae Protein Tyrosine Phosphatase, PTP1, was expressed from an Escherichia coli expression system and milligram quantities of active PTP1 were purified chromatographically. The substrate specificity of the recombinant PTP1 was probed using synthetic phosphotyrosine-containing peptides corresponding to the regulatory phosphorylation sites of the yeast MAP kinase homologues $Fus3_{176-186}$, $Kss1_{179-189}$, and $Hog1_{170-180}$. Peptide sequences derived from the MAP kinase homologues were chosen arbitrarily as starting points for sequence variation studies even though they are not likely to be candidates for physiological substrates of PTP1. Phosphotyrosyl-$Hog1_{170-180}$ peptide showed a $K_M$ value of 877 ${\mu}M$ and phosphorylated $Kss1_{179-189}$ and $Fus3_{176-186}$ peptides showed lower $K_M$ values of 74 ${\mu}M$ and 51 ${\mu}M$ each. To study the effect of sequence variations of the peptide, amino acids of the undecapeptide $Hog1_{170-180}$ (DPQMTGpYVSTR) were sequentially substituted by an alanine residue. More extensive variations of each amino acid revealed positional importance of each amino acid residue. Based on these results, we derived a peptide sequence (DADEpYDA) that is recognized by PTP1 with an affinity ($K_M$ is 4 ${\mu}M$) significantly higher than that of the peptides derived from the phosphorylation sites of Fus3, Kss1, and Hog1.

• Journal of Microbiology and Biotechnology
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• v.14 no.1
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• pp.171-177
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• 2004

The endoxylanase (642 bp; 213 amino acids) and $\beta$-xylosidase (1,602 bp; 533 amino acids) genes from Bacillus sp. were amplified by PCR and separately inserted into the downstream of the yeast ADH1 promoters, resulting in the pAEDX-1 (7.63 kb) and pAEX (8.47 kb) plasmids, respectively. When the yeast transformants, S. cerevisiae SEY2102 harboring pAEDX-1 or pAEX, were grown on YPD medium, the total activities of the enzymes were approximately 9.8 unit/ml for endoxylanase and 2.9 unit/m1 for $\beta$-xylosidase. When the three kinds of xylan from oat spelts, birch wood, and corncob were hydrolyzed by treating with recombinant endoxylanase and $\beta$-xylosidase, it was found that xylose, xylobiose, and xylotriose were produced. To efficiently hydrolyze xylan, various reaction conditions such as amount of enzymes, substrate type, substrate concentration, temperature, and reaction time were examined. The optimized conditions for the hydrolysis of xylan were as follows: amount of endoxylanase, 10 units; amount of $\beta$-xylosidase, 10 units; temperature, $50^\circ{C}$; substrate type, oat spelts xylan; substrate concentration, 6%; reaction time, 1 h. Under the optimal condition, xylose was mainly produced from oat spelts xylan by cooperative action of endoxylanase and $\beta$-xylosidase.