• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2001년도 추계학술발표대회
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• pp.89-92
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• 2001

Aspergillus niger has been used as a host system to express many heterologous proteins. It has various advantages over other expression systems in that it is a small eukaryotic GRAS (Generally Recognized aS Safe) organism with a capacity of secreting large amount of foreign proteins. However, it has been known that the presence of an abundant protease is a limiting factor to express a heterologous protein. The proteases deficient mutants of A. niger were obtained using UV -mutagenesis. A total of 1 ${\times}$ $10^5$ spores were irradiated with 10-20% survival dose of UV, 600J/M2 at 280nm, and the resulting spores were screened on the casein -gelatin plates. Ten putative protease deficient mutants were further analyzed on the starch plates to differentiate the pro from the secretory mutant. An endogenous extracellular enzyme, glucose oxidase, was also examined to confirm that the mutant phenotype was due to the proteases deficiency rather than the mutation in the secretory pathway. The reduced proteolytic activity was measured using SDS-fibrin zymography gel, casein degradation assay, and bio-activity of a supplemented hGM -CSF (human Granulocyte-Macrophage Colony Stimulating Factor). Comparing with the wild type strain, less than 30 % of proteolytic activity was observed in the culture filtrate of the protease deficient mutant (pro -20) without any notable changes in cell growth and secretion.

• 한국미생물·생명공학회지
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• 제17권4호
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• pp.273-276
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• 1989

Bti 균주를 배양하여 포자와 $\delta$-endotoxin의 복합물을 직접 field에 적용시키면 미생물 생태계에 변화를 초래할 가능성이 있으며, 배양 후 포자로부터 $\delta$-endotoxin만을 분리할 경우에는 대량생산을 위한 기술적인 어려움이 있다. 또한 $\delta$-endotoxin은 용혈성 단백질을 함유하고 있어 Bti 균주로부터 무포자이며 $\delta$-endotoxin을 생산하는 변이주의 분리와 아울러 $\delta$-endotoxin의 용혈성 인자의 제거 또는 약화를 시도하였다. 그 결과 Bti 균주를 열처리시켜 SPO3 (Spo$^-$ Cry$^+$) 변이주를 분리 선별하고, 그 $\delta$-endotoxin의 모기유충에 대한 LC$_{50}$ 및 응혈성을 조사해 본 결과 LC$_{50}$는 약 20배, 용혈성은 약 25배정도 야생주의 $\delta$-endotoxin에 비해 약화되었다. 이는 변이주의 $\delta$-endotoxin내에 28KDa 단백질의 소실에 기인된 것이라 생각되며, 이는 면역확산반응과 면역전기영동 및 SDS-PAGE에 의해 확인되었다.

• BMB Reports
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• 제31권3호
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• pp.287-295
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• 1998

Acetolactate synthase (ALS) is the first common enzyme in the biosynthesis of L-Ieucine, L-isoleucine, and L-valine. The sulfonylurea-resistant ALS gene from Nicotiana tabacum was cloned into the bacterial expression vector pGEX-2T. The resulting recombinant plasmid pGEX-ALS3 was used to transform Escherichia coli strain XL1-Blue, and the mutant tobacco ALS (mALS) was expressed in the bacteria as a protein fused with glutathione S-transferase (GST). The fusion product GST-mALS was purified in a single step on a glutathione-Sepharose column. ALS activities of 0.9-2.5 ${\mu}mol/min/mg$ protein were observed in the GST-mALS, and the Km values for pyruvate, FAD, and TPP were 10.8-24.1, $(1.9-8.9){\times}10^{-3}$, and 0.14-0.38 mM, respectively. The purified GST-mALS was resistant to both the sulfonylurea and the triazolopyrimidine herbicides, and lost its sensitivity to end products, L-valine and L-leucine. For comparision, the tobacco wild-type recombinant ALS fused with GST, GST-wALS, was also characterized with respect to its pyruvate and cofactor bindings. These results suggest that the purified mutant recombinant tobacco ALS was functionally active, that the mutations resulting in herbicide resistance has affected pyruvate and cofactor bindings," and that the two classes of herbicides interact at a common site on the plant ALS.

• Molecules and Cells
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• 제19권2호
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• pp.256-261
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• 2005

In silico analysis of genome of the cyanobacterium Synechocystis sp. PCC 6803 identified two genes, slr0329 and sll0593, that might participate in glucose (Glc) phosphorylation (www.kazusa.or.jp/cyano). In order to determine the functions of these two genes, we generated deletion mutants, and analyzed their phenotypes and enzymatic activities. In the presence of 10 mM Glc, wild-type (WT) and slr0329 defective strain (M1) grew fast with increased respiratory activity and NADPH production, whereas the sll0593 deletion mutant (M2) failed to show any of the Glc responses. WT and M1 were not significantly different in their glucokinase activity, but M2 had 90% less activity. Therefore, we propose that Sll0593 plays a major role in the phosphorylation of glucose in Synechocystis cells.

• 한국미생물·생명공학회지
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• 제24권4호
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• pp.408-414
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• 1996

The SecY is a central component of the protein export machinery that mediate the translocation of secretory proteins across the plasma membrane, and has been known to be rate-limiting factor of secretion in Escherichia coli. In order to study the extracellular protein secretion in Gram-positive microorganism, we have, constructed strains harboring more than one copy of the gene for SecY. Firstly, the gene, for B. subtilis SecY and its promoter region was subcloned into pDH32 and the chimeric vector was inserted into amyE locus by homologous recombination. Secondly, low copy number vector, pCED6, was also used for subcloning the secY gene and for constructing a strain which harbors several copies of secY. The KH1 cell which harbor two copies of secY on the chromosome excreted more extracellular proteins than the wild type PB2. Moreover, the KH2 cells which harbor several copies of secY in pCED6 vector excreted more extracellular proteins than the KH1 cells. Here, we found that the capacity of protein secretion is partly controlled by the number of secY and it is suggested that SecY has also an important role in protein secretion in B. subtilis, a gram positive microorganism, as like in E. coli. This will promote the use of B. subtilis as a host for the expression of useful foreign gene and excretion of precious proteins.

• 대한수의학회지
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• 제58권3호
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• pp.137-141
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• 2018

The efficacy of the CA-2-MP120 vaccine, a cell culture-attenuated strain of virulent porcine reproductive and respiratory syndrome virus (PRRSV), was assessed in pigs. Despite the persistence of viremia in all vaccinated animals during the immunization period, the virus was not detected in vaccinated pigs following challenge. Furthermore, no pigs in the vaccinated group shed PRRSV nasally, orally or rectally throughout the experiment. Moreover, histopathological lung and lymph node lesions in the immunized group were much milder than those in the unimmunized and challenged group. These results indicated that CA-2-MP120 can provide effective protection against virulent wild-type PRRSV-2.

• BMB Reports
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• 제28권3호
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• pp.216-220
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• 1995

cDNA for human cholesteryl ester transfer protein (CETP), a potent atherogenic plasma protein that redistributes the neutral lipids among lipoproteins, was expressed in recombinant vaccinia virus-infected cells (CV-1). Two insertion vectors regulated by different promoters were constructed. The vectors were introduced into human thymidine kinase-negative ($TK^-$) 1438 cells infected with wild-type vaccinia virus (WR strain). Recombinant viruses were selected with 5-bromodeoxyuridine (BUdR) and X-gal and identified with DNA dot blot analysis (vSC11-CETP and vTM1-CETP). The CETP cDNA insert in the recombinant vaccinia virus genome was identified by Southern blot analysis. Transcription of CETP cDNA in CV-1 cells infected with recombinant vaccinia virus was monitored by Northern blot analysis using the CETP cDNA as a probe. Positive signals were detected at 1.8 kb in cells infected with vSC11-CETP and at 2.3 kb in cells infected with vTM1-CETP. The recombinant vaccinia virus-infected CV-1 cells were shown to produce functional CETP when the culture medium was subjected to the CETP assay.

• 환경생물
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• 제22권4호
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• pp.487-493
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• 2004

To investigate the antifungal activity related protein in pesticidal bacteria, a bacterial strain LTD was isolated from soil collected at Gimje in Jeonbuk province, Korea, and identified as Bacillus subtilis LTD based on a API50 CHB kit and 168 rDNA sequencing. It has an antifungal activity against 9 plant pathogenic fungi in a paper disc assay. The antifungal activity- deficient mutant, B. subtilis mLTD was induced at a 5 kGy dose of $^{60}Co$ gamma radiation. Using the two-dimensional electrophoresis and the matrix assisted laser desorption ionization time-of-flight mass spectrometry, the comparison analysis of proteins between the wild and mutant were performed. A major intracellular serine proteinase IspA (MW: 32.5 kDa), a NAD (P) H dehydrogenase (MW: 20.0 kDa), and a stage II sporulation protein AA, SpoIIAA (MW: 14.3kDa) were detected only in the B. subtilis LTD. These results suggested that the functions of these proteins found only in the B. subtilis LTD could. be closely related to the antifungal activity against plant pathogenic fungi.

• The Plant Pathology Journal
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• 제25권3호
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• pp.205-212
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• 2009

Colletotrichum acutatum was the main cause of the recent outbreaks of anthracnose on pepper fruit in Korea. To facilitate molecular analysis of C. acutatum, we generated an arginine auxotrophic mutant of the C acutatum strain JC24 using a targeted gene replacement strategy. A 3.3-kb genomic region carrying an ortholog (designated CaARG2) of the fungal gene encoding N-acetylglutamate synthase, the first enzyme of arginine biosynthesis in fungi, was deleted from the fungal genome. The mutant exhibited normal growth only when arginine was exogenously supplied into the culture medium. Transformation of the arginine auxotrophic mutant with a plasmid DNA carrying an intact copy of CaARG2, which was smaller than the deleted region in the mutant, not only caused random vector insertions in the fungal genome, but also recovered both hyphal growth and pathogenicity of the mutant to the wild-type level. Using this new selection system, we have successfully developed a restriction enzyme-mediated integration procedure, which would provide an economically efficient random mutagenesis method in C. acutatum.

• 한국잠사곤충학회지
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• 제39권1호
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• pp.22-29
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• 1997

Ultraviolet weavelength (UV) of 366 nm produced clearer fluorescent dolor than that of 254 nm for the inspection of silkworm cocoons. Fluorescent color of silkworm cocoons varied in color, appears no relationship with the natural color under the normal light. Uniformity of fluorescent color was improved by selection of blue or yellow line from wild types. Blue and yellow, located at the opposite poles on the color solid and L*a*b* color system, confirmed as pure standard of fluorescent color in the silkworm races for commercial white cocoons. the cocoons with blue fluorescence occupied as high as 1.7 to 8.6 times than those with yellow in the Japanese silkworm races. Fluorescence of silkworm cocoon was not affected by forced flow dry at 70$^{\circ}C$ for 6 hrs. While the Japanese races revealed no sexual difference in fluorescent color, sex-dependence of the color was common in the Chinese races for commercial white cocoon. The fluorescence of cocoon shell of Chinese races showed clear separation of blue of median color. Silkworm strain of Dc20 and Fc24 were sexualy segregated 98.8${\pm}$1.20%, 99.0${\pm}$1.00% by cocoon fluorescence, as that of 99.3${\pm}$0.44% by typical larval marking of sex-limited inheritance. Specific expression of cocoon fluorescence, applicable to breeding of simple discrimination of sex for Chinese races, inspected thoroughly on the surface and inner layer of cocoon shell.