• Korean Journal of Pharmacognosy
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• v.37 no.1 s.144
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• pp.21-27
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• 2006

Yeast based estrogenicity assay is the simplest and useful for the assay and the discovery of novel estrogenic substances in natural specimens, The estrogen receptor(ER) modulation activity of 50% EtOH extracts of 101 traditional medicinal herbs was assessed using a recombinant yeast assay system with both a human estrogen receptor expression plasmid and a receptor plasmid. Among them, 14 species proved to be active. Pureariae Flos (flower of Puerraria thunbergiana BENTH.) had the highest estrogenic relative potency$(7.75{\times}10^{-3})$ $(EC_{50}=9.39\;{\mu}g/ml)$. The $EC_{50}$ value of $17{\beta}-estradiol$ used as the positive control was $0.073\;{\mu}g/ml)$ (Relative Potency=1.00). There results demonstrated that some of the traditional medical herb may be useful in the therapy of estrogen replacement.

• BMB Reports
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• v.38 no.6
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• pp.683-689
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• 2005

Antibody to hepatitis B surface antigen (HBsAb) is the important serological marker of the hepatitis B virus (HBV) infection. Conventionally, the hepatitis B surface antigen (HBsAg) obtained from the plasma of HBV carriers is used as the diagnostic antigen for detection of HBsAb. This blood-origin antigen has some disadvantages involved in high cost, over-elaborate preparation, risk of infection, et al. In an attempt to explore the suitable recombinant HBsAg for the diagnostic purpose, the HBV S gene was expressed in Pichia pastoris and the product was applied for detection of HBsAb. Hepatitis B virus S gene was inserted into the yeast vector and the expressed product was analyzed by sodium dodecyl sulphate polyacrolamide gel electrophoresis (SDS-PAGE), immunoblot, electronic microscope and enzyme linked immunosorbent assay (ELISA). The preparations of synthesized S protein were applied to detect HBsAb by sandwich ELISA. The S gene encoding the 226 amino acid of HBsAg carrying ahexa-histidine tag at C terminus was successfully expressed in Pichia pastoris. The His-Tagged S protein in this strain was expressed at a level of about 14.5% of total cell protein. Immunoblot showed the recombinant HBsAg recognized by monoclonal HBsAb and there was no cross reaction between all proteins from the host and normal sera. HBsAb detection indicated that the sensitivity reached 10 mIu (micro international unit)/ml and the specificity was 100% with HBsAb standard of National Center for Clinical Laboratories. A total of 293 random sera were assayed using recombinant S protein and a commercial HBsAb ELISA kit (produced by blood-origin HBsAg), 35 HBsAb positive sera and 258 HBsAb negative sera were examined. The same results were obtained with two different reagents and there was no significant difference in the value of S/CO between the two reagents. The recombinant HBV S protein with good immunoreactivity and specificity was successfully expressed in Pichia pastoris. The reagent for HBsAb detection prepared by Pichia pastoris-derived S protein showed high sensitivity and specificity for detection of HBsAb standard. And a good correlation was obtained between the reagent produced by recombinant S protein and commercial kit produced by blood-origin HBsAg in random samples.

• Microbiology and Biotechnology Letters
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• v.17 no.3
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• pp.263-268
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• 1989

To maximize the performance of recombinant cell fermentation process through optimizing environmental conditions, the production of invertase from recombinant Saccharomyces cerebisiae Containing SUC2 gene was studied as a model. The recombinant cells showed biphasic growth on glucose. Since the promoter of the SUC2 is regulated by the concentration of glucose in the medium, expression of invertase by recombinant yeast began when the glucose concentration decreased in a range of 0.25-0.4 g/L during the batch culture. Plasmid segregation occured frequently during glucose fermentation, and infrequently during ethanol oxidation. A rapid appearance of invertase activity with glucose was observed under nonaerated condition, and the maximum specific invertase activity was about 1.5 times as high as under aerobic condition, In fed batch culture, when n low level of glucose was continuously supplied to the tormentor after the time of glucose depletion during growth phase, specific and total invertase activity increased about 1.7 and 2.9 fold, respectively, in a batch culture.

• Journal of Microbiology
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• v.44 no.5
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• pp.515-522
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• 2006

A cDNA library of Ganoderma lucidum has been constructed using a Zap Express cloning vector. A glyceraldehyde-3-phosphate dehydrogenase gene (gpd) was isolated from this library by hybridization of the recombinant phage clones with a gpd-specific gene probe generated by PCR. By comparison of the cDNA and the genomic DNA sequences, it was found that the complete nucleotide sequence encodes a putative polypeptide chain of 338 amino acids interrupted by 6 introns. The predicted amino acid sequence of this gene shows a high degree of sequence similarity to the GPD proteins from yeast and filamentous fungi. The promoter region contains a CT-rich stretch, two CAAT boxes, and a consensus TATA box. The possibility of using the gpd promoter in the construction of new transformation vectors is discussed.

• 한국생물공학회:학술대회논문집
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• 2000.04a
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• pp.478-481
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• 2000

The INU2 gene encoding an endoinulinase of Aspergillus ficuum was expressed by the Kluyveromyces marxianus INU1 promoter in a SUC2-deleted Saccharomyces cerevisiae to produce the endoinulinase free of an exoinulinase and an extracellular invertase in the culture medium. When inulin was included in the medium, a recombinant yeast strain produced the sufficient amount of the enzyme to make a halo around its colony. An expression of endoinulinase was dependent on the culture temperature and shaking. The highest expression of endoinulinase was observed at $30^{\circ}C$, and 150rpm.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.333-335
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• 2001

A simple control strategy of DO-stat was introduced to the recombinant rGuamerin production process in Pichia pastoris. This induction strategy consisted of two interrelated control loops ‘by which oxygen ratio of inlet gas and methanol feeding rate was controlled. Using this control strategy, over-feeding or under-feeding of methanol could be avoided in concomitance with the efficient control of dissolved oxygen level. As a result, the cell concentration reached 130 g/L and rGuamerin expression level was 450 iu/L, which was more than 40% increased result comparing with the fed-batch process using manual control of methanol feeding rate.

• Journal of Microbiology and Biotechnology
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• v.11 no.1
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• pp.61-64
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• 2001

Delta-integration vectors were constructed for the purpose of achieving homologous integration of the hirudin expression cassette into the chromosome of Saccharomyces cerevisiae. A double $\delta$ system truncated with the unnecessary bacterial genes, and consequently having a reduced insert size for integration, showed a four-fold increase in transformation efficiency at given DNA concentrations, and as a result, the constructed recombinant yeast strain had a 1.3-fold enhancement in hirudin expression level compared with a single $\delta$ system.

• 한국생물공학회:학술대회논문집
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• 2000.11a
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• pp.376-379
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• 2000

The methylotrophic yeast Pichia pastoris is one of the best host for the production of foreign proteins because of the presence of the strong AOX1 promoter induced by methanol. Methanol feeding induces the protein production and provides energy sources for the host cells. However, excess methanol inhibits the growth of host cells, while an insufficient methanol lead to poor growth and protein production. We have used various controled methanol feeding strategies to obtain the maximum proteins.

• 한국생물공학회:학술대회논문집
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• 2000.11a
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• pp.669-671
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• 2000

Antimicrobial cationic peptides have attracted increasing research and clinical interest as a natural antibiotics due to their broad spectrum of antimicrobial activites and the rapid development of multidrug-resistant pathogenic microorganisms. In this study, first, we synthesized artificial fusion partner and cationic peptide genes (lactoferricin, magainin, protegrin-1, and indolicidin). Second, we constructed recombinant expression vectors and then transformed Pichia pastoris. Finally, expressed cationic peptides were purified and tested for their antimicrobial activites. Antimicrobial activity has been tested upon the appearance of clearing zone on the plate with the lawn of gram negative E.coli XL- I blue and garm positive Staphylococcus aureus. Protegrin-1 and Indolicidin have apparant activity of cationic peotides. This fusion technique may lead to a general and suitable tool for production of pure antimicrobial cationic peptides in Pichia pastoris.

• Biomolecules & Therapeutics
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• v.9 no.3
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• pp.162-166
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• 2001

Helicobacter pylori is a major cause of gastric-associated diseases. In our previous study, the Adhesin/CTXA2B was expressed as insoluble recombinant chimeric protein derived from the H. pylori adhesin genetically coupled to CTXA2B subunit in Escherichia coli. Since it is very important to optimize IPTG concentration, culture temperature and composition of medium to maximize cell growth and productivity, these conditional growth factors were determined for increasing the productivity of the expressed Adhesin/CTXA2B chimeric protein in Escherichia coli JM101 carrying pTEDhpa/ctxa2b. Our data demonstrate that optimal medium for increased production of chimeric protein was a YCP/Glu medium composed of 2% yeast extract, 1% casamino acid, phosphate solution [0.3% $KH_2P0_4$, 0.4% $Na_2HP0_4$, 0.25% ($NH_4)_2HPO_4$], and 0.5% glucose. In addition, optimal concentration of IPTG was 1 mM and culture temperature, $37^{\circ}C$.