• Korean Journal of Microbiology
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• v.31 no.3
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• pp.203-207
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• 1993

Five different secretion vectors were constructed by varying the signal sequences and .alpha.-antitrypsin (.alpha.1-AT) a numan secretory protein, was produced from yeast cells. The signal sequences used are those of acid phosphatase (PH05) and .alpha.-factor (M f.alphal1) of Saccharomyces cerevisiae, glucoamylase (STA1) of Saccharomyces diastaticus, and human .alpha.1-AT. Four vectors directed the efficient secretion of .alpha.1-AT ito the culture media. The secretion vector carrying the glucoamylase signal sequence (pGAT11) showed the highest efficiency of secretion. About 70% of .alpha.1-AT produce dwere secreted into the media. The endo H treatment of partially purified .alpha.1-AT indicates that the secreted .alpha.1-AT appeared to be glycosylated. This glycosylation pattern was altered when amino acid substitution mutations were introduced at the three glycosylation sites of .alpha.-AT.

• Bulletin of the Korean Chemical Society
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• v.35 no.9
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• pp.2781-2786
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• 2014

Human ${\alpha}_1$-antitrypsin (${\alpha}_1$-AT) is a natural inhibitor of neutrophil elastases and has several dozens of genetic variants. Most of the deficient genetic variants of human ${\alpha}_1$-AT are unstable and cause pulmonary emphysema. However, the most clinically significant variant, Z-type ${\alpha}_1$-AT, exhibits retarded protein folding that leads to accumulation of folding intermediates. These aggregate within the endoplasmic reticulum (ER) of hepatocytes, subsequently causing liver cirrhosis as well as emphysema. Here, we studied the role of an ER folding assistant protein Cpr5p on Z-type ${\alpha}_1$-AT folding. Cpr5p was induced > 2-fold in Z-type ${\alpha}_1$-AT-expressing yeast cells compared with the wild type. Knockout of CPR5 exacerbated cytotoxicity of Z-type ${\alpha}_1$-AT, and re-introduction of CPR5 rescued the knockout cells from aggravated cytotoxicity caused by the ${\alpha}_1$-AT variant. Furthermore, Cpr5p co-immunoprecipitated with Z-type ${\alpha}_1$-AT and facilitated its protein folding. Our results suggest that protein-folding diseases may be suppressed by folding assistant proteins at the site of causal protein biosynthesis.

• Journal of Microbiology
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• v.41 no.4
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• pp.335-339
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• 2003

Many genetic variants of ${\alpha}_1$-antitrypsin have been associated with early onset emphysema and liver cirrhosis. However, the detailed structural basis of pathogenic ${\alpha}_1$-antitrypsin molecules is rarely known. Here we found that a recombinant $M_{malton}$ variant (Phe52-deleted) lost inhibitory activity by spontaneous conformational conversion into a more stable, inactive form under physiological conditions. Biochemical and spectroscopic data suggested that the variant converts into a reactive center loop-inserted conformation, resembling the latent form of plasminogen activator inhibitor-1.

• Proceedings of the Korean Biophysical Society Conference
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• 1996.07a
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• pp.14-14
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• 1996

The metastable native state of proteins plays an important role in regulating biological functions. The native strain of serpins (serine protease inhibitors) are considered to be crucial for the inhibitory function. Several thermostable mutations of human $\alpha$$_1$-antitrypsin, a prototype inhibitory serpin, were identified in a systematic search targeted at the hydrophobic core of the molecule [Nature structural biology, vol. 3, no. 6, 497-500(1996)]. (omitted)

• Proceedings of the Korean Biophysical Society Conference
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• 1996.07a
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• pp.13-13
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• 1996

Aggregation of human $\alpha$$_1$-antitrypsin ($\alpha$$_1$-AT) during folding occurs both in vitro and in vivo. In vivo aggregates of mutant $\alpha$$_1$-AT such as $M_{malton}$ (Phe52 deleted) and Z (Glu342 longrightarrowLys) variants have pathological consequences. In order to analyze the process of $\alpha$$_1$-AT aggregation in detail, the folding-unfolding kinetics of $\alpha$$_1$-AT was examined by monitoring intrinsic Trp fluorescence and ANS binding. (omitted)

• Proceedings of the Korean Biophysical Society Conference
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• 2001.06a
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• pp.31-31
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• 2001

The native strain of serpins (serine protease inhibitors) has been recognized as a mechanism of biological regulation. Indeed, some stabilizing single residue mutations of human $\alpha$$_1$-antitrypsin, a prototype serpin, relieved local strain and caused the loss of inhibitory activity. The native strain of $\alpha$$_1$-antitrypsin is distributed throughout the whole molecule, but the strain that regulates the function directly is highly localized in the regions that appear to be mobilized during complex formation with a target protease.(omitted)

• Proceedings of the Korean Biophysical Society Conference
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• 1997.07a
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• pp.39-39
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• 1997

Native strain of inhibitory SERPINS (Serine protease inhibitors) is thought to be used in the facile conformational switch to play biological regulation. Many heat stable variants of $\alpha$$_1$-antitrypsin, a prototype of inhibitory serpins, increased their stability by reducing the native strain.(omitted)

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.74-74
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• 2002

Production of u 1-antitrypsin ($\alpha$AT) in transgenic cows has a great value in the field of medicine. The present study was conducted to determine the effect of chemically defined KSOM media on in vitro development of bovine transgenic nuclear transfer (NT) embryos. An expression plasmid for human $\alpha$AT was constructed by inserting a bovine beta-casein promoter, a green fluorescent protein (GFP) marker gene, and a human $\alpha$AT target gene into a pcDNA3 plasmid. Cumulus cells as donor nuclei in NT were collected from a Holstein cow and transfected by lipid-mediated method using FuGene6 (Roche Molecular Biochemicals, USA) as reagent. GFP expressed cumulus cells were introduced into recipient oocytes under DIC microscopy equipped with FITC filter set. After electrical fusion and chemical activation, reconstructed embryos were cultured in 1) SOF + 0.8% BSA, 2) KSOM + 0.8% BSA, 3) KSOM + 10% FBS and 4) KSOM +0.01% PVA for 192 h at 39$^{\circ}C$ with 5% $CO_2$, 5% $O_2$ and 90% $N_2$in humidified condition. The development of the embryos was recorded and the GFP expression in blastocyst was determined under FITC filter. The average fusion rate was 73.8% (251/340; n=8). The development rates to 2-4 cells, morula, blastocysts and expression rates in blastocysts varied from 70.3 to 76.5%, 30.2 to 33.8%, 25.4 to 33.8% and 11.8 to 15.6%, respectively. The difference in development and expression rates of embryos among 4 culture groups was not significant (P>0.05). This study indicates that chemically defined KSOM medium is also able to support development of bovine transgenic NT embryos at similar rate of SOF or KSOM supplemented with BSA or serum.

• Proceedings of the Zoological Society Korea Conference
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• 1995.10b
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• pp.80-80
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• 1995

No Abstract, See Full Text

• Korean Journal of Microbiology
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• v.30 no.2
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• pp.108-112
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• 1992

To investigate the expression and the secretion of heterologous proteins in yeast, we constructed an yeast secretion vector and produced a human secretory protein, .alpha.-1-antitrypsin (.alpha.-1-AT), from yeast cells. The secretion vector pGAT8 was constructed by inserting the signal sequence of yeast acid phosphatase gene (PH05) into the .alpha.1-AT expression vector pGAT6 which contained .alpha.-1-AT cDNA fused to GAL10-CYC1 promotor. The .alpha.-1-AT was produced efficiently in the yeast cells transformed with plasmid pGAT8, which was onfirmed both by the .alpha.-1-AT activity assay and by the immunoblot method using .alpha.-1-AT antibody. We also showed the secretion of .alpha.-1-AT into the culture media and into the periplasmic space by immunoblot.