• Microbiology and Biotechnology Letters
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• v.29 no.4
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• pp.206-211
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• 2001

In order to overexpress constitutively the Kluyveromyces marxianus exoinulinase gene (INUI) in Saccharomyces cerevisiae, four episomal expression systems employing GAPDH, ADHI, PGK and ENOI promoters were constructed as p YIGP aADHI -INU, pPGK-INU, and pENOI- INU plasmids respectively, When S cereviais transformants harboring each plasmid were batchwisely cultivated in the fermentor containing 5% glucose medium no significant differences in the cell growth are observed How- ever the experession level of exoinulinase and plasmid stability showed a strong dependency on the promoter employed. The expression levels of exoinulinase were about 1.70 unit/ml for GAPDH promoter 1.67 unit/ml for PGK promoter, 1.29 unit /ml for ADH1 promoter, and 0.80 unit/ml for ENOl promoter. The plasmid stabilites were maintaines above 80% in all experession systems. except the GAPDH promoter system of 55%, Based on the plas- mid stability and expression level of exoinulinase the ADHl and PGK promoter system were selected for the fed - batch culture to overproduce exoinulinase By the intermittent feeding of yeast extract and glucose, both promoter systems gave the cell concentration of about 30 g-dry cell weight/1 byt the maximal exoinulinase activity of 3.70 unit/ml and plasmid stability of 96% in the ADH1 promoter were higher than those (2.70 unit/ml, 80%) of PGK sys- tem Taking into account the plasmid stability and extended culture time the ADH1 promoter systems would be the most feasible expression systems for the constitutive overproduction of exoinulinase through high cell-density fed- batch cultures using non-selective rich medium.

• KSBB Journal
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• v.16 no.1
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• pp.15-23
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• 2001

The development of expression systems for heterologous proteins has been greatly demanded not only for the study of the structure/function relationships of these proteins but also for their biotechnological and pharmaceutical applications. During the past decades, the methylotrophic yeast Hansenula polymorpha and Pichia pastoris have drawn attention as one of promising hosts for the production of a variety of heterologous proteins. The increasing popularity of H. polymorpha and P. pastoris as the host systems can be attributed to the several advantages over the traditional yeast Saccharomyces cerevisiae, such as the availability of very strong and tightly regulated promoters from the enzymes involved in the metabolism of methanol, a very high-cell density even on simple mineral media, and a high stability of expression plasmids. Furthermore, it has been observed that glycoproteins from these two yeasts are less hyperglycoylated compared to those from S. cerevisiae. Despite substantial similarities as methylotrophic yeasts, however, these two expression systems have some unique features distinguished from each other. In this paper we present a brief overview on the present status of the expression systems developed in methylotrophic yeast, mainly focusing on the similarities and differences between the H. polymorpha and P. pastoris systems.

• KSBB Journal
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• v.22 no.4
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• pp.185-190
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• 2007

Productivity of secreted recombinant protein depends largely on its stability in the extracellular environment with protease. Most hGM-CSF produced by transgenic tobacco cell cultures and secreted to the medium was confirmed to be rapidly degraded by protease in medium. To increase the productivity, therefore, various protein stabilizers such as gelling agents such as carrageenan and alginate, polymers, polyols, and amino acids have been tested. The stability of hGM-CSF in spent medium without cells was improved by the presence of gelling agents. However, the reason for the enhanced production by the addition of gelling agents may be due to the increased expression level and permeability rather than stability. The addition of DMSO inhibited the cell growth, but improved specific yield. The others were not effective for stability as well as hGM-CSF production.

• Journal of People, Plants, and Environment
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• v.22 no.4
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• pp.365-373
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• 2019

This study was conducted to determine the effects of horticultural therapy on self-esteem of women with hearing impairment. Ten women with hearing impairment registered in the Mokpo Branch of the Korean Association of the Deaf participated in the horticultural therapy programs (HTP) for self-esteem. The self-esteem scale was used to evaluate the effects of the programs and scores were compared to determine the difference before and after the programs. Self-esteem was improved in all 10 subjects, and its mean value increased with statistical significance from 22.5 points before the programs to 29.8 points after the programs. The programs preferred by women with hearing impairment included 'making a centerpiece using scented candles and flowers', 'packing and planting pot spray chrysanthemum', 'making a topiary', 'planting Hedera helix', and 'planting cyclamen'. Therefore, it was suggested that self-esteem of women with hearing impairment were improved by increasing social and family support, emotional stability, positive feedback, and self-expression through horticultural therapy programs.

• Journal of Microbiology and Biotechnology
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• v.10 no.3
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• pp.321-326
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• 2000

To examine the feasibility of the long-term production of the human granulocyte colony stimulating factor (hG-CSF) using the $_{L}-arabinose$ promoter system of Escherichia coli, flask relay culture and cyclic fed-batch culture were performed. In the flask relay culture, it was found that the pismid was maintained stably up to about 170 generations in an uninduced condition, whereby the cells could also maintain the capability of expressing hG-CSF expression were maintained stably up to at least 100 generations. In contrast, in the cyclid fed-batch culture, segregational plasmid instability was observed within about 4 generations after induction, even though the cell growth and hG-CSF production reached their maximum balues, 78.0 g/l of dry cell weight and 7.0 g/l of hG-CSF, respectively. It would appear that, when compared to the flask relay culture, the high-cell density and high-level expression of hG-CSF in the cyclic fed-batch cultrure led to the segregational plasmid instability; in other words, a severe metabolic burden existe on the cells due to the high-level expression of hG-CSF. Accordingly, based on these long-term cultures, the segregational and structural plasmid instability was observed and a strategy to overcome such problems could be designed.

• Fisheries and Aquatic Sciences
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• v.22 no.12
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• pp.28.1-28.8
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• 2019

Background: In the present study, we evaluated four commonly used housekeeping genes, viz., actin-β, elongation factor-1α (EF1α), acidic ribosomal protein (ARP), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as internal references for quantitative analysis of immune genes in nervous necrosis virus (NNV)-infected seven-band grouper, Hyporthodus septemfasciatus. Methods: Expression profiles of the four genes were estimated in 12 tissues of healthy and infected seven-band grouper. Expression stability of the genes was calculated using the delta Ct method, BestKeeper, NormFinder, and geNorm algorithms. Consensus ranking was performed using RefFinder, and statistical analysis was done using GraphpadPrism 5.0. Results: Tissue-specific variations were observed in the four tested housekeeping genes of healthy and NNV-infected seven-band grouper. Fold change calculation for interferon-1 and Mx expression using the four housekeeping genes as internal references presented varied profiles for each tissue. EF1α and actin-β was the most stable expressed gene in tissues of healthy and NNV-infected seven-band grouper, respectively. Consensus ranking using RefFinder suggested EF1α as the least variable and highly stable gene in the healthy and infected animals. Conclusions: These results suggest that EF1α can be a fairly better internal reference in comparison to other tested genes in this study during the NNV infection process. This forms the pilot study on the validation of reference genes in Hyporthodus septemfasciatus, in the context of NNV infection.

• Preventive Nutrition and Food Science
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• v.5 no.4
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• pp.184-188
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• 2000

HMG-CoA reductase is th rate-limiting enzyme of cholesterol biosynthesis. As intracellular levels of cholesterol should be regulated elaborately in response to external stimuli an internal needs, the expression of the HMG-CoA reductase gene is regulated intricately at several different levels from transcription to post-translational modification. In this study, we investigated the regulatory mechanism of HMG-CoA reductase gene expression at the post-transcriptional/pre-translational levels in a baby hamster kidney cell line, C100. when 25-hydroxycholesterol was added to cells cultured in medium containing 5% delipidized fetal bovine serum and 25$\mu$M lovastatin, the levels of HMG-CoA reductase mRNA decreased rapidly, which seemed to be due to the increased degradation of reductase mRNA. These suppressive effects of 25-hydroxycholesterol on MG-CoA reductase mRNA levels were blocked by a translation inhibitor, cycloheximide. Similarly, actinomycin D and 5,6-dichloro-1-$\beta$-D-ribofuranosylbenzimidazole, transcription inhibitors, blocked the 25-hydroxycholesterol-mediated degradation of HMG-CoA reductase mRNA. These results indicate that new protein/RNA synthesis is required for the degradation of HMG-CoA reductase mRNA. In addition, data from the transfection experiments shows that cis-acting determinants, regulating the stability of reductase mRNA, were scattered in the sequence corresponding to 1766-4313 based on the sequence of Syrian hamster HMG-CoA reductase cDNA. Our data suggests that sterol-mediated destabilization of reductase mRNA might be one of the important regulatory mechanism of HMG-CoA reductase gene expression.

• Korean Chemical Engineering Research
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• v.56 no.6
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• pp.826-831
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• 2018

This study presents fabrication of bi-compartmental particles labeled by multiple fluorescence. To compartmentalize fluorescent expression at the particle, two fluorescent dyes with less overlap of the excitation and emission spectra are selected. To ensure the fluorescence stability, the fluorescent dyes contain acrylate functional groups in the molecules so that they can be cross-linked together with monomers constituting the particle. Strong fluorescent expression and compartmentalization were observed at the particle fabricated using the selected fluorescent dyes through confocal microscopy. Furthermore, long-term fluorescence stability was verified by measuring fluorescent expression and intensity for 4 weeks. We anticipate that the bi-compartmental particles labeled by multiple fluorescence can be widely used for multi-target drug delivery system, analysis of 3 dimensional Brownian motion, and investigation of 3 dimensional complex self-assembled morphologies.

• Molecules and Cells
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• v.42 no.5
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• pp.426-439
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• 2019

Many small RNAs (sRNAs) regulate gene expression by base pairing to their target messenger RNAs (mRNAs) with the help of Hfq in Escherichia coli. The sRNA DsrA activates translation of the rpoS mRNA in an Hfq-dependent manner, but this activation ability was found to partially bypass Hfq when DsrA is overproduced. The precise mechanism by which DsrA bypasses Hfq is unknown. In this study, we constructed strains lacking all three rpoS-activating sRNAs (i.e., ArcZ, DsrA, and RprA) in $hfq^+$ and $Hfq^-$ backgrounds, and then artificially regulated the cellular DsrA concentration in these strains by controlling its ectopic expression. We then examined how the expression level of rpoS was altered by a change in the concentration of DsrA. We found that the translation and stability of the rpoS mRNA are both enhanced by physiological concentrations of DsrA regardless of Hfq, but that depletion of Hfq causes a rapid degradation of DsrA and thereby decreases rpoS mRNA stability. These results suggest that the observed Hfq dependency of DsrA-mediated rpoS activation mainly results from the destabilization of DsrA in the absence of Hfq, and that DsrA itself contributes to the translational activation and stability of the rpoS mRNA in an Hfq-independent manner.

• BMB Reports
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• v.32 no.4
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• pp.345-349
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• 1999

We fused the promoter region of an H3.2 chicken histone gene, whose expression is dependent on the cell cycle, to the 5' coding region of an H3.3 chicken histone gene, which is expressed constitutively at a low level throughout the cell cycle. This fusion gene showed a cell cycle-regulated pattern of expression, but in a different manner. The mRNA level of the fusion gene increase during the S phase of the cell cycle by about 3.7-fold at 6 h and 2.7-fold at 12 h after the serum stimulation. The mRNA level of the intact H3.2 gene, however, increased by an average of 3.6-fold at 6 h and 8.7-fold at 12 h. This different expression pattern might be due to the differences in their 3' end region that is responsible for mRNA stability. The 3' end of the H3.2 mRNA contains a stem-loop structure, instead of a poly(A) tail present in the H3.3 mRNA. We also constructed a similar fusion gene using a H3.3 histone gene whose introns had been eliminated to rule out the possibility of involvement of the introns in cell cycle-regulated expression. The expression of this fusion gene was almost identical to the fusion gene made previously. These results indicate that the promoter region of the H3.2 gene is only partially responsible for its expression during the S phase of the cell cycle.