• Animal cells and systems
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• 제3권2호
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• pp.181-185
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• 1999

Xenopus oocyte is one of the widely used heterologous expression systems of ion channels for electrophysiological studies. Here we describe a new method in which cRNA produced by polymerase chain reaction (PCR) and in vitro transcription is injected to express ion channels in oocytes. This method enables us (1) to eliminate all or a part of the untranslated region of the cDNA and to replace it with a known sequence which helps increase the expression level in oocytes, and (2) to use the PCR product for in vitro transcription without subcloning. Using this method, the expression level of one of the neuronal nicotinic acetylcholine receptors (nAChRs) $\alpha$$_{6}$ subtype in oocytes was systematically increased by more than 100-fold, which was confirmed both by the $\alpha$-Bungarotoxin ($\alpha$,/TEX>Bgt) binding assay and the current measurement.t.

• Molecules and Cells
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• 제27권5호
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• pp.601-608
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• 2009

The 2-deoxystreptamine and paromamine are two key intermediates in kanamycin biosynthesis. In the present study, pSK-2 and pSK-7 recombinant plasmids were constructed with two combinations of genes: kanABK, and kanABKF and kacA respectively from kanamycin producer Streptomyces kanamyceticus ATCC12853. These plasmids were heterologously expressed into Streptomyces lividans TK24 independently and generated two recombinant strains named S. lividans SK-2/SL and S. lividans SK-7/SL, respectively. ESI/ MS and ESI-LC/MS analysis of the metabolite from S. lividans SK-2/SL showed that the compound had a molecular mass of 163 $[M+H]^+$, which corresponds to that of 2-deoxystreptamine. ESI/MS and MS/MS analysis of metabolites from S. lividans SK-7/SL demonstrated the production of paromamine with a molecular mass of $324[M+H]^+$. In this study, we report the production of paromamine in a heterologous host for the first time. This study will evoke to explore complete biosynthetic pathways of kanamycin and related aminoglycoside antibiotics.

• 미생물학회지
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• 제30권2호
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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.

• Journal of Microbiology and Biotechnology
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• 제23권10호
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• pp.1413-1421
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• 2013

The marine microalga Isochrysis sphaerica is rich in the very-long-chain polyunsaturated fatty acids, including eicosapentaenoic acid (EPA, $C20:5{\omega}-3$) and docosahexaenoic acid (DHA, $C22:6{\omega}-3$) that are important to human health. Here, we report a functional characterization of a ${\Delta}4$-fatty acid desaturase gene (FAD4) from I. sphaerica. IsFAD4 contains a 1,284 bp open reading frame encoding a 427 amino acid polypeptide. The deduced amino sequence comprises three conserved histidine motifs and a cytochrome b5 domain at its N-terminus. Phylogenetic analysis indicated that IsFad4 formed a unique Isochrysis clade distinct from the counterparts of other eukaryotes. Heterologous expression of IsFAD4 in Pichia pastoris showed that IsFad4 was able to desaturate docosapentaenoic acid (DPA) to form DHA, and the rate of converting DPA to DHA was 79.8%. These results throw light on the potential industrial production of specific polyunsaturated fatty acids through IsFAD4 transgenic yeast or oil crops.

• Molecular & Cellular Toxicology
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• 제5권4호
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• pp.283-290
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• 2009

Differential gene expression profiling was performed in the hepatic tissue of marine medaka fish (Oryzias javanicus) after exposure to benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH), by heterologous hybridization using a medaka cDNA microarray. Thirty-eight differentially expressed candidate genes, of which 23 were induced and 15 repressed (P<0.01), were identified and found to be associated with cell cycle, development, endocrine/reproduction, immune, metabolism, nucleic acid/protein binding, signal transduction, or non-categorized. The presumptive physiological changes induced by BaP exposure were identified after considering the biological function of each gene candidate. The results obtained in this study will allow future studies to assess the molecular mechanisms of BaP toxicity and the development of a systems biology approach to the stress biology of organic chemicals.

• 대한바이러스학회지
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• 제28권2호
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• pp.193-201
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• 1998

In Sprague Dawley (SD) rats, antibodies against strains of Orientia tsutsugamushi, Kato, Karp and Gilliam, were produced in order to investigate their longevity and cross-reactivities to their corresponding homologous and heterologous antigens. By immunofluorescence assay (IFA) of IgG and IgM, it was shown that the immunity to the homologous strains persisted at a higher level (longevity of at least 34 weeks with higher IFA titers). On the other hand, the immunity to the heterologous strains persisted at a lower level (longevity of 10 to 34 weeks with lower IFA titers). Since infection with one strain of O. tsutsugamushi does not preclude reinfection with other strains, understanding of the antigenic diversity of O. tsutsugamushi and duration of the immunity to both homologous and heterologous strain is very important in diagnosis of scrub typhus.

• 한국동물학회지
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• 제33권3호
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• pp.303-309
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• 1990

대장균에서 진핵세포의 펩타이드 호르몬 전구체를 대량으로 생산할 목적으로 T7 overexpression system을 이용하여 angler 어류의 prepro-SRIF I 유전자와 T7 phage coat 단백질 S10 유전자를 결합시켜 일련의 융합유전자를 합성하였다. 이 융합유전자를 가지고 있는 숙주 대장균, BL21 DE3는 3가지 종류의 서로 다른 SRIF 관련 폴리펩타이드를 대량 합성하였다. 본 연구에서는 대량합성된 폴리펩타이드의 특성을 규명하였으며, 펩타이드 호르몬 전구체를 얻기 어려운 이종이에서의 대량발현의 중요성과 표적 펩타이드 호르몬인 SRIF의 적용성을 논의하였다.

• Journal of Microbiology and Biotechnology
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• 제18권5호
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• pp.866-873
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• 2008

2-Deoxystreptamine is a core aglycon that is vital to backbone formation in various aminoglycosides. This core structure can be modified to develop hybrid types of aminoglycoside antibiotics. We obtained three genes responsible for 2-deoxystreptamine production, neo7, neo6, and neo5, which encode 2-deoxy-scyllo-inosose synthase, L-glutamine: 2-deoxy-scyllo-inosose aminotransferase, and dehydrogenase, respectively, from the neomycin gene cluster. These genes were cloned into pIBR25, a Streptomyces expression vector, resulting in pNDOS. The recombinant pNDOS was transformed into a non-aminoglycoside-producing host, Streptomyces venezuelae YJ003, for heterologous expression. Based on comparisons of the retention time on LC-ESI/MS and ESI-MS data with those of the 2-deoxystreptamine standard, a compound produced by S. venezuelae YJ003/pNDOS was found to be 2-deoxystreptamine.

• Journal of Animal Science and Technology
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• 제63권2호
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• pp.405-416
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• 2021

The aim of the study was to determine the effect of whole milk powder (WMP) as heterologous proteins on chicken breast emulsion-type sausages. The quality properties of WMP on such chicken breast emulsion-type sausages were investigated by measuring the proximate composition, pH, color, cooking yield, protein solubility, and by applying other methods, such as texture profile analysis (TPA), microphotograph, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and electronic nose. The crude fat, protein, and ash contents of 15% WMP samples were significantly higher than the control samples (p < 0.05). The redness of the cooked samples significantly increased with an increase in the WMP contents (p < 0.05). The cooking yield of WMP treated samples was significantly higher than the control sample (p < 0.05). Additionally, the hardness, gumminess, and chewiness of WMP treated samples were significantly higher than the control sample (p < 0.05). The sarcoplasmic and myofibrillar proteins of samples containing 15% WMP were significantly higher than the control samples (p < 0.05). The result of SDS-PAGE showed that the C protein, sarcoplasmic protein, actin, and tropomyosin increased with an increase in the WMP contents. The principal component analysis plot of WMP-treated samples was clearly different from that of the control samples. Based on these results, it was predicted that WMP could be useful as heterologous protein on emulsion-type sausage.

• Journal of Microbiology and Biotechnology
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• 제31권11호
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• pp.1465-1480
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• 2021

Violacein, a purple pigment first isolated from a gram-negative coccobacillus Chromobacterium violaceum, has gained extensive research interest in recent years due to its huge potential in the pharmaceutic area and industry. In this review, we summarize the latest research advances concerning this pigment, which include (1) fundamental studies of its biosynthetic pathway, (2) production of violacein by native producers, apart from C. violaceum, (3) metabolic engineering for improved production in heterologous hosts such as Escherichia coli, Citrobacter freundii, Corynebacterium glutamicum, and Yarrowia lipolytica, (4) biological/pharmaceutical and industrial properties, (5) and applications in synthetic biology. Due to the intrinsic properties of violacein and the intermediates during its biosynthesis, the prospective research has huge potential to move this pigment into real clinical and industrial applications.