• Asian-Australasian Journal of Animal Sciences
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• v.27 no.9
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• pp.1355-1359
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• 2014

To detect goat vascular endothelial growth factor (VEGF)-mediated regrowth of hair, full-length VEGF164 cDNA was cloned from Inner Mongolia cashmere goat (Capra hircus) into the pET-his prokaryotic expression vector, and the recombinant plasmid was transferred into E. coli BL21 cells. The expression of recombinant $6{\times}his-gVEGF164$ protein was induced by 0.5 mM isopropyl thio-${\beta}$-D-galactoside at $32^{\circ}C$. Recombinant goat VEGF164 (rgVEGF164) was purified and identified by western blot using monoclonal anti-his and anti-VEGF antibodies. The rgVEGF164 was smeared onto the dorsal area of a shaved mouse, and we noted that hair regrowth in this area was faster than in the control group. Thus, rgVEGF164 increases hair growth in mice.

• Journal of Microbiology and Biotechnology
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• v.10 no.2
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• pp.119-136
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• 2000

Being autotrophic, algae occupy a trategic place in the biosphere. They produce oxygen both directly and indirectly through the chloroplasts of all green plants. The chloroplasts are believed to have originated from archaic prokaryotic algae through endosymbiosis with primitive eukaryotic cells. Phytoplankton and other algae regulate the global environment not only by releasing oxygen but also by fixing carbon dioxide. They affect water quality, help in the treatment of sewage, and produce biomass. They can be used to produce hydrogen which is a clean fuel, and biodiesel, and fix $N_2$ for use as a biofertilizer. Some other services of algae to the environment include restoration of metal damaged ecosystems, reducing the atmospheric $CO_2$ load and citigating global warming, reclamation of saline-alkaline unfertile lands, and production of dimethyl sulphide (DMS) and oxides of nitrogen (NOx) involved in the regulation of UV radiation. ozone concentration, and global warming. Algae can be valuable in understanding and resolving certain environmental issues.

• The Microorganisms and Industry
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• v.19 no.4
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• pp.27-31
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• 1993

Certain bacterial species possess the capability of differentiation through several morphogenetic changes which enable them to adapt to certain internal and external stimuli(Losick and Shapiro 1984). Upon induction, cells of A. vinelandii undergo a morphological process which leads to the production of one cyst per cell (Sadoff, 1975). The cysts are considerably resistant to desiccation, which confers a survival advantages upon the organism(Socolofsky and Wyss 1962). Like other prokaryotic differentiations encystment provides a relatively simple model of cellular differentiation. Like in other differentiating bacteria, vegetative growth can be separated from differentiation. Furthermore, the differentiation cycle can be synchronized by specific inducer. There have been a great deal of morphological and physiological studies on this process. However, the mechanisms used to regulate cell differentiation can be clearly defined by careful genetic analysis of the process. Unfortunately, A. vinelandii has proven to be difficult for genetic analysis (Sadoff 1975). For example, it has been shown that a variety of metabolic mutants of Azotobacter speicies are difficult to isolate after mutagenesis with chemical mutagens or UV irradiation. Nevertheless recent advances in molecular genetics in Azotobacter species, especially in the nitrogen fixation research area, appear to be able to overcome this difficulty (Robinson et al. 1986; Kennedy et al. 1986).

• Journal of Species Research
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• v.11 no.1
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• pp.1-9
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• 2022

The phylum Actinobacteria includes many groups of aerobic, Gram-stain-positive, rod, or filamentous shaped bacteria. Actinobacteria are known for multicellular differentiation in some groups, and also for production of various secondary metabolites such as antibiotics. During a series of extensive surveys of indigenous prokaryotic species diversity in Korea, bacterial strains belonging to Actinobacteria were isolated from various sources of terrestrial environments. A total of 21 bacterial strains, belonging to 10 genera in 8 families, were isolated as unrecorded species in Korea. Among them, 11 were assigned to the family Streptomycetaceae, two species assigned to each of the families Microbacteriaceae, Mycobacteriaceae and Nocardioidaceae, and one species assigned to each of the families Euzebyaceae, Corynebacteriaceae, Micrococcaceae and Intrasporangiaceae. At the genus level, Streptomyces (10 species) was the most abundant, followed by Microbacterium and Mycolicibacterium(2 species each), and one species in each of the genera Corynebacterium, Euzebya, Arthrobacter, Terracoccus, Kribbella, Nocardioides and Yinghuangia. The detailed descriptions of each unrecorded species are provided.

• Journal of Microbiology and Biotechnology
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• v.25 no.7
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• pp.1007-1014
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• 2015

Plectasin, the first defensin extracted from a fungus (the saprophytic ascomycete Pseudoplectania nigrella), is attractive as a prospective antimicrobial agent. The purpose of this study was to establish a bacterium-based production system and evaluate the antimicrobial activity of the resulting plectasin. A gene encoding plectasin, with the codon preference of Escherichia coli, was optimized based on its amino acid sequence, synthesized using genesplicing with overlap extension PCR, and inserted into the expression vector pGEX-4T-1. The fusion protein was expressed in the soluble fraction of E. coli and purified using glutathione Stransferase affinity chromatography. Plectasin was cleaved from the fusion protein with thrombin and purified by ultrafiltration. The purified plectasin showed strong, concentrationdependent antimicrobial activity against gram-positive bacteria, including antibiotic-resistant bacteria, especially penicillin-resistant Enterococcus faecium. This antimicrobial activity was equal to chemically synthesized plectasin and was maintained over a wide range of pH and temperatures. This soluble recombinant expression system in E. coli is effective for producing plectasin at a relatively lower cost, and higher purity and efficiency than prior systems, and might provide a foundation for developing a large-scale production system. Overall, plectasin shows potential as a novel, high-performance, and safe antibiotic for the treatment of refractory diseases caused by drug-resistant bacterial strains.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.69.1-69
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• 2003

RSH (relA/spoT homolog) has been known to determine the level of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), which are the effector nucleotide of the prokaryotic stringent response and also play a role in antibiotic production and differentiation in Streptomyces species but not a little in eukaryotic organism, especially in plant. Salicylic acid (SA), a critical signal molecule of establishing systemic acquired resistance (SAR), could induce SAR in Pepper (Capcicum annuum) against Phytophthora capsici. And the extent of SAR induction was in proportion to the dosage of SA (or BTH). Suppression subtractive hybridization (SSH), a PCR-based method for cDNA subtraction, was carried out between SA-treated and non-SA-treated pepper leaves to isolate genes which may be responsible for defense signaling against pathogens. Early upregulated gene was selected from reverse northern and kinetics of SSH-genes transcripts in SA-treated pepper leaves upon SA treatment. Full-length cDNA of the gene (PepRSH； Pepper RelA / SpoT homolog) had an open reading frame (ORF) of 2166 bp encoding a protein of 722 amino acids and a significant homology with (p)ppGpp phosphohydrolase or synthetase. Genomic DNA gel blot analysis showed that pepper genome has at least single copy of PepRSH. PepRSH transcripts was very low in untreated pepper leaves but strongly induced by SA and methyljasmonic acid (MeJA), indicating that PepRSH may share common SA and MeJA-mediated signal transduction pathway Functional analysis in E. coli showed PepRSH confers phenotypes associated with (p)ppGpp synthesis through a complementation using active site mutagenesis.

• Animal cells and systems
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• v.15 no.1
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• pp.29-36
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• 2011

Insect lysozymes are basic, cationic proteins synthesized in fat body and hemocytes in response to bacterial infections and depolymerize the bacterial cell wall. The c-type lysozyme of the insect Spodoptera litura (SLLyz) is a single polypeptide chain of 121 residues with four disulfide bridges and 17 rare codons and is approximately 15 kDa. The full-length SLLyz cDNA is 1039 bp long with a poly(A) tail, and contains an open reading frame of 426 bp long (including the termination codon), flanked by a 54 bp long 5' UTR and a 559 bp long 3' UTR. As a host for the production of high-level recombinant proteins, E. coli is used most commonly because of its low cost and short generation time. However, the soluble expression of heterologous proteins in E. coli is not trivial, especially for disulfide-bonded proteins. In order to prevent inclusion body formation, GST was selected as a fusion partner to enhance the solubility of recombinant protein, and fused to the amplified products encoding mature SLLyz. The expression vector pGEX-4T-1/rSLLyz was then transformed into E. coli BL21(DE3)pLysS for soluble expression of rSLLyz, and the soluble fusion protein was purified successfully. Inhibition zone assay demonstrated that rSLLyz showed antibacterial activity against B. megaterium. These results demonstrate that the GST fusion expression system in E. coli described in this study is efficient and inexpensive in producing a disulfide-bonded rSLLyz in soluble, active form, and suggest that the insect lysozyme is an interesting system for future structural and functional studies.

• Journal of Life Science
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• v.9 no.3
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• pp.253-261
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• 1999

Leptin gene, an obesity gene, has been known to involve in the regulation of food intake and body weight. It is also thought to be related to the glucose metabolism, insulin secretion and type II diabetes mellitus. Recently, the production of recombinant leptin protein has been attempted for the application in the treatment of obesity and the correction of hereditary obesity and type II diabetes. In the present study, leptin cDNA was cloned from mouse fat cells by RT-PCR and prokaryotic expression of leptin was attempted in order ot prepare a leptin-specific antigen. Immunization of a rabbit with the leptin-specific antigen into a rabbit resulted in the generation of leptin-specific antiserum that could be useful in the detection of leption expressed in various tissues. The sequence of leptin cDNA prepared in the present study wa identical to the previously reported one. Transformation of E. coli(DH5a) cells with the leptin cDNA-inserted translation vector, pGEX-4T-3-leptin followed by treatment with IPTG (0.1mM) resulted in the expression of a large amount of GST-leptin fusion protein with a molecular weight of 44 KDa as an inclusion body. Denaturation of the insoluble fusion protein by 8M urea, 6M guanidium-HCI or 0.1% 2-mercaptoethanol followed by a slow oxidation could not solubilize the inclusion body. The cell extract was subjected to SDS-PAGE and GST-leptin protein electroeluted from the gel was then injected into a rabbit subcutaneously for the immunization. Anti-GST-leptin rabbit antiserum which had a cross reactivity to the GST-leptin protein was generated. Leptin protein expressed in mouse brain and fat tissues was detected by Western blot immunodetection system using the antiserum generated in the present study.

• Journal of Microbiology and Biotechnology
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• v.17 no.5
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• pp.728-732
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• 2007

In the present article, a novel fusion expression vector for Escherichia coli was developed based on the pTORG plasmid, a derivative of pET32a. This vector, named pT7MT(GenBank Accession No DQ504436), carries a T7 promoter and it drives the downstream gene encoding Metallothionein 2A(MT2A). There are in-framed multiple cloning sites(MCS) downstream of the MT2A gene. A target gene can be cloned into the MCS and fused to the C-terminal of the MT2A gene in a compatible open reading frame(ORF) to achieve fusion expression. The metal-binding capability of MT2A allows the purification of fusion proteins by metal chelating affinity chromatography, known as $Ni^{2+}$-affinity chromatography. Using this expression vector, we successfully got the stable and high-yield expression of MT2A-GST and MT2A-Troponin I fusion proteins. These two proteins were easily purified from the supernatant of cell lysates by one-step $Ni^{2+}$-affinity chromatography. The final yields of MT2A-GST and MT2A-Troponin I were 30mg/l and 28mg/l in LB culture, respectively. Taken together, our data suggest that pT7MT can be applied as a useful expression vector for stable and high-yield production of fusion proteins.

• The Journal of the Convergence on Culture Technology
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• v.10 no.3
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• pp.873-878
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• 2024

We collected rat feces by group period after oral administration of fructooligosaccharides and fruit and vegetable complex extracts for 2 weeks in the Sprague-Dawley rat model of loperamide-induced constipation and analyzed trends in changes in the intestinal microbiome. Microbial composition analysis was performed on Fractoologosaccharide and fruit and vegetable complex extracs(FVCE), by 16S rDNA cloning and pyrosequencing to obtain basic data for the standardization and systematization of the FVCE manufacturing process. Microbial analysis of the prokaryotic community revealed a slight difference in microbial verrucomicrobiota was dominant at the phylum level. At the genus level, prevotella and muribaculaceae showed further differences at the species level. These results suggest that the microbial community used affects the quality of fruit and vegetable complex extracs(FVCE) produced. Thus, a stable microbial community must be maintained for the production of fruit and vegetable complex extracs(FVCE) with consistent quality.