Discovery of cSNPs in Pig Using Full-length Enriched cDNA Libraries of the Korean Native Pig as a Source of Genetic Diversity

Dirisala, Vijaya R.,Kim, Ju-Hyun,Park, Kwang-Ha,Lee, Hoon-Taek,Park, Chan-Kyu Korean Society for Biotechnology and Bioengineerin 2007 Biotechnology and Bioprocess Engineering Vol.12 No.4

Clones from full-length enriched cDNA libraries serve as valuable resources for functional genomic studies. We analyzed 3,210 chromatograms obtained from sequencing the 5'-ends of brainstem, liver, neocortex, and spleen clones derived from full-length enriched cDNA libraries of Korean native pigs. In addition, 50,000 pig EST sequence trace files were obtained from Genbank and combined with our sequencing information for SNP identification in silica. For the SNP analysis, neocortex, and liver libraries were newly constructed, whereas the sequencing results from brainstem and spleen libraries were from previously constructed libraries. The putative SNPs from the in silica analysis were confirmed by genomic PCR from a group of 20 pigs of four different breeds. Using this approach, 86% of cSNPs identified in silico were confirmed and the SNP detection frequency was 1 SNP per 338 bp. Interestingly, we found a valine deletion at amino acid position 126 of the neuronal and endocrine protein gene in the Korean native pig. We confirmed that this deletion was caused by alternative splicing at the NAGNAG acceptors. Our study shows that large-scale EST sequencing of Korean native pigs can be effectively employed for natural polymorphism-based pig genome analysis.

Surface Displayed Expression of a Neutralizing Epitope of Spike Protein from a Korean Strain of Porcine Epidemic Diarrhea Virus

Park, Seung-Moon,Mo, Ae-Young,Lim, Jung-Gu,Chung, Hea-Jong,Kim, Tae-Geum,Kim, Kang-Ju,Cho, Dong-Ha,Yang, Moon-Sik,Kim, Dae-Hyuk Korean Society for Biotechnology and Bioengineerin 2007 Biotechnology and Bioprocess Engineering Vol.12 No.6

The neutralizing epitope (K-COE) of the spike protein from a Korean strain of porcine epidemic diarrhea virus (PEDV) has been shown to prevent and foster an immune response to PED, when orally adjusted. The cell surface of the budding yeast, Saccharomyces cerevisiae, was engineered to anchor the K-COE on the outer layer of the cell, and consequently, the altered yeast was applied as a dietary complement for animal feed, with immunogenic functions. In this study, the K-COE gene (K-COE) of the Korean strain of PEDV with the signal peptide of rice amylase 1A (Ramy1A), was fused with the gene encoding the carboxyterminal half (320 amino acid residues from the C terminus) of yeast ${\alpha}-agglutinin$, a mating associated protein that is anchored covalently to the cell wall. The glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter was selected in order to direct the expression of the fusion construct, and the res 내 ting recombinant plasmid was then introduced into S. cerevisiae. The surface display of K-COE was visualized via confocal microscopy using a polyclonal antibody against K-COE as the primary antibody, and FITC (fluorescein isothiocyanate)-conjugated goat anti-mouse IgG as the secondary antibody. The display of the K-COE on the cell surface was further verified via Western blot analysis using the cell wall fraction after the administration of ${\alpha}-1,3-glucanase/PNGase\;F/{\beta}-mannosidase$ treatment.

Development of a Serial Bioreactor System for Direct Ethanol Production from Starch Using Aspergillus niger and Saccharomyces cerevisiae

Jeon, Bo-Young,Kim, Soo-Jin,Kim, Dae-Hee,Na, Byung-Kwan,Park, Doo-Hyun,Tran, Hung Thuan,Zhang, Ruihong,Ahn, Dae-Hee Korean Society for Biotechnology and Bioengineerin 2007 Biotechnology and Bioprocess Engineering Vol.12 No.5

Aspergillus niger hyphae were found to grow with unliquefied potato starch under aerobic conditions, but did not grow under anaerobic conditions. The raw culture of A. niger catalyzed saccharification of potato starch to glucose, producing approximately 12 g glucose/L/day. The extracellular enzyme activity was decreased in proportion to incubation time, and approximately 64% of initial activity was maintained after 3 days. At $50^{\circ}C$, A. niger hyphae growth stopped, while the extracellular enzyme activity peaked. On the basis of the A. niger growth property and enzyme activity, we designed a serial bioreactor system composed of four different reactors. Fungal hyphae were cultivated in reactor I at $30^{\circ}C$, unliquefied starch was saccharified to glucose by a fungal hyphae culture in reactors II and III at $50^{\circ}C$, and glucose was fermented to ethanol by Saccharomyces cerevisiae in reactor IV. The total glucose produced by fungal hyphae in reactor I and saccharification in reactor II was about 42 g/L/day. Ethanol production in reactor IV was approximately 22 g/L/day, which corresponds to about 79% of the theoretical maximum produced from 55 g starch/L/day.

Use of L-Buthionine Sulfoximine for the Efficient Expression of Disulfide-containing Proteins in Cell-free Extracts of Escherichia coli

Oh, In-Seok,Kim, Tae-Wan,Ahn, Jin-Ho,Keum, Jung-Won,Choi, Cha-Yong,Kim, Dong-Myung Korean Society for Biotechnology and Bioengineerin 2007 Biotechnology and Bioprocess Engineering Vol.12 No.5

We have developed a technique to improve the formation of correct disulfide bonds within cell-free synthesized proteins. Via the use of a metabolic inhibitor of glutamate-cysteine ligase, the accumulation of glutathione was effectively prevented in cell-free extracts, thereby enabling the stable maintenance of redox potential for extended reaction periods. As a result, in a reaction in which a model protein contatining 9 disulfide bonds was synthesized under cell-free conditions, the final amount of active protein products was increased by 50%. The method presented in this study will provide a rapid and robust route to the high-throughput expression and screening of proteins which require multiple disulfide bonds for their activity.

Effects of Temperature Shift Strategies on Human Preproinsulin Production in the Fed-batch Fermentation of Recombinant Escherichia coli

Son, Young-Jin,Park, Kyong-Hee,Lee, Sang-Yong,Oh, Sung-Jin,Kim, Chang-Kyu,Choi, Byoung-Taek,Park, Yong-Cheol,Seo, Jin-Ho Korean Society for Biotechnology and Bioengineerin 2007 Biotechnology and Bioprocess Engineering Vol.12 No.5

Preproinsulin is a well-known precursor of human insulin for the regulation of blood glucose levels. In this study, fed-batch fermentations of recombinant Eschericha coli JM109/pPT-MRpi were carried out for the overexpression of human preproinsulin. The expression of human preproinsulin was controlled by the temperature inducible P2 promoter. The time-course profiles of fed-batch fermentation and SDS-PAGE analysis showed that human insulin expression was triggered by a culture temperature change from 30 to $37^{\circ}C$. Fermentation shift strategies, including the multi-step increase of temperature and the modulation of initiation time, were optimized to obtain high titers of cell mass and preproinsulin. The optimized fed-batch fermentation, consisting of a three-step shift of culture temperature from 30 to $37^{\circ}C$ for 2 h, gave the best results of 43.1 g/L of dry cell weight and 33.3% preproinsulin content, which corresponded to 2.0- and 1.2-fold increases, respectively, as compared to those of fed-batch culture at a constant temperature of $37^{\circ}C$.

Antibacterial Activity of Ulva lactuca against Methicillin-Resistant Staphylococcus aureus (MRSA)

Kim, In-Hae,Lee, Dong-Gun,Lee, Sang-Hyun,Ha, Jong-Myung,Ha, Bae-Jin,Kim, Sung-Koo,Lee, Jae-Hwa Korean Society for Biotechnology and Bioengineerin 2007 Biotechnology and Bioprocess Engineering Vol.12 No.5

The in vitro antimicrobial activity of the marine green algae Ulva lactuca was examined against gram-positive bacteria, gram-negative bacteria, and a fungus. The ethyl-ether extract of algae exhibited a broad-spectrum of antibacterial activity, but not antifungal activity against Candida albicans. In particular, the U. lactuca extract showed strong activity against the bacterium methicillin-resistant Staphylococcus aureus (MRSA). This result confirms the potential use of seaweed extracts as a source of antibacterial compounds or as a health-promoting food for aquaculture.

Aflatoxins: Detection, Toxicity, and Biosynthesis

Do, Jin-Hwan,Choi, Dong-Kug Korean Society for Biotechnology and Bioengineerin 2007 Biotechnology and Bioprocess Engineering Vol.12 No.6

Aflatoxins are toxic and carcinogenic secondary metabolites produced mainly by Aspergillus flavus and Aspergillus parasiticus. The aflatoxins present in food and feed are hazardous to both human and animal health. A number of studies have been conducted on the detection, toxicity, biosynthesis, and regulation of aflatoxins due to the discovery of serious aflatoxicosis in farm animals, and the presence of aflatoxins in many food products. There are many reviews that focus on the biosynthesis of aflatoxin, yet there are few examinations of the overall aspects of aflatoxins, including detection, toxicity, and the regulation of biosynthesis. Thus, the goal of this article is to give: an overview of the overall aspects of aflatoxins. This review consists of four parts: i) detection methods for aflatoxins, ii) the toxicity mechanism of aflatoxin B1, iii) gene cluster for aflatoxin biosynthesis, and iv) the regulation of aflatoxin biosynthesis.

Increased hGM-CSF Production and Secretion with Pluronic F-68 in Transgenic Nicotiana tabacum Suspension Cell Cultures

Cho, Jong-Moon,Kwon, Jun-Young,Lim, Jung-Ae,Kim, Dong-Il Korean Society for Biotechnology and Bioengineerin 2007 Biotechnology and Bioprocess Engineering Vol.12 No.6

The effects of Pluronic F-68, a nonionic surfactant, on the production and secretion of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) in a transgenic Nicotiana tabacum cell suspension culture were investigated in this study. The addition of Pluronic F-68 was shown to extend cell survival in the stationary phase, but had no influence on effective initial cell growth. With regard to production, it increased the level of extracellular hGM-CSF two-fold. This may be attributable not only to the enhanced expression level, but also to the improved permeability of the cell membrane due to the interaction between Pluronic F-68 and the cell membrane and cell wall. The effect of Pluronic F-68 on the production and secretion of hGM-CSF in a bioreactor was also evaluated. hGM-CSF production in the bioreactor after the addition of Pluronic F-68 proved more effective than in flask cultures.

Effects of Mechanical Stimulation on the Proliferation of Bone Marrow-derived Human Mesenchymal Stem Cells

Choi, Kyung-Min,Seo, Young-Kwon,Yoon, Hee-Hoon,Song, Kye-Yong,Kwon, Soon-Yong,Lee, Hwa-Sung,Park, Jung-Keug Korean Society for Biotechnology and Bioengineerin 2007 Biotechnology and Bioprocess Engineering Vol.12 No.6

To support and enhance the in vitro growth and activity of mesenchymal stem cells (MSCs), the cell culture medium may be supplemented with various proteins and factors to mimic the physiological environment in which the cells optimally proliferate and differentiate. In this study, the effects of mechanical factors on cellular metabolic responses were investigated experimentally using a bioreactor. The effects of various chemical factors, such as growth factors, cytokines, and hormones, were also investigated. Based on previous reports demonstrating the important roles of mechanical factors in the growth and activity of MSCs, we sought to evaluate the effects of mechanical stimuli on the proliferation of bone marrow-derived MSCs using a cell training bioreactor that imposed cyclic mechanical stretch, with parameters of 240 min/day, 0.03 Hz, and $5{\sim}15%$ strain. The application of cyclic stretch $(5{\sim}15%\;strain)$ to the MSCs enhanced their proliferation during the early stage(3 days), but not the late stage (14 days), of batch culture. Mechanical stretch did not increase the release of lactate dehydrogenase (LDH) from the MSCs during culture. Appropriate levels of mechanical stretch $(5{\sim}10%\;strain)$ increased collagen synthesis, but did not alter MSC stretch was able to serve as a potent positive modulator of MSC proliferation during the initial stages of culture.

Induction and Purification of a Thermophilic Chitinase Produced by Aeromonas sp. DYU-Too7 Using Glucosamine

Lien, T.S.,Yu, S.T.,Wu, S.T.,Too, J.R. Korean Society for Biotechnology and Bioengineerin 2007 Biotechnology and Bioprocess Engineering Vol.12 No.6

In the presence of chitin, Aeromonas sp. DYU-Too7 can produce extra-cellular, chitin-degrading enzymes. Chitin analogues and other carbon sources can be used to cultivate this bacterial strain. The chitinases produced by the strain were higher in the GIcN (glucosamine) medium than those in other media. The maximal chitinase activity occurred in the medium containing 0.1% GlcN. Cultivation of Aeromonas sp. DYU-Too7 in the GlcN medium sped up the chitinase production; however, the same result did not appear when it was cultivated in the (Chitin + GlcN) medium. This result may indicate that GlcN can be utilized by Aeromonas sp. DYU-Too7 as a carbon source and an inducer to produce chitinases. A chitinase with a molecular mass of 36 kDa was further purified and characterized to have an optimal reacting pH of 5.0 and an optimal reacting temperature of $50^{\circ}C$. This chitinase showed high stability in the proximity of $30^{\circ}C$ and also high stability in the proximity of pH 7.0. The hydrolysates of colloidal chitin, with the aid of the 36-kDa chitinase, were analyzed by an HPLC and found to be chitobiose.