• BMB Reports
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• 제33권3호
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• pp.256-262
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• 2000

In this study the disassembly process of chlorophyII (ChI)protein complexes of a stay-green mutant (ore10 of Arabidopsis thaliana) was investigated during the dark incubation of detached leaves. During this dark-induced senescence (DIS), the Chi loss was delayed in the mutant, while the photochemical efficiency of photosystem II (PSII) or Fv/Fm was accelerated when compared with the wild type (WT) leaves. This indicates that the decrease in Fv/Fm is a separate process and not causally-linked to the degradation of Chi during DIS of Arabidopsis leaves. In the native green gel electrophoresis of the Chi-protein complexes, which was combined with an additional twodimensional SDS-PAGE analysis, the delayed senescence of this mutant was characterized by the appearance of an aggregate at 1 d or 2 d, as well as very stable light harvesting complex II (LHCII) trimers until 5 d after the start of DIS. The polypeptide composition of the aggregates varied during the whole DIS at 5 d. Dl protein appeared to be missing in the aggregates. This result supports the idea of a faster depletion of functional PSH in the mutants compared with WT, as suggested by the earlier reduction of Fv/Fm and the stable Chl a/b ratio in the mutants. At 5 d, the WT leaves also often showed aggregates, but the polypeptide composition was different from those of ore10. The results presented suggest that the formation of aggregates, or stable LHCII trimers in the stay-green mutants, is a way to structurally protect Chi-protein complexes from serious proteolytic degradation. Detailed disassembly processes of Chi-protein complexes in WT and ore10 mutants are discussed.

• Journal of Microbiology and Biotechnology
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• 제13권6호
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• pp.949-954
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• 2003

The argG gene of Corynebacterium glutamicum encoding argininosuccinate synthetase (EC6345) was cloned and sequenced. The gene was cloned by heterologous complementation of an Escherichia coli arginine auxotrophic mutant (argG/sup -/). The cloned DNA fragment also complements E. coli argD, argF, and argH mutants, suggesting a clustered organization of the genes in the chromosome. The coding region of the argG gene is 1,206 nucleotides long with a deduced molecular weight of about 44 kDa, comparable with the predicted size of the expressed protein on the SDS-PAGE. Computer analysis revealed that the amino acid sequence of the argG gene product had a high similarity to that of Mycobacterium tuberculosis and Streptomyces clavuligerus. Two conserved sequence motifs within the ArgG appear to be ATP-binding sites which correspond to 2 of the 3 conserved regions found in sequences of all known argininosuccinate synthetases.

• Journal of Microbiology and Biotechnology
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• 제23권11호
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• pp.1554-1559
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• 2013

A protease produced by Bacillus polyfermenticus SCD was purified and characterized as a new detergent material. The protease was purified from supernatant produced by B. polyfermenticus SCD, by ammonium sulfate precipitation, ion-exchange chromatography on a DEAE-Sephadex A-50, and finally gel filtration chromatography on Sephadex G-50. The molecular mass of this enzyme was 44 kDa based on SDS-PAGE. The optimum temperature and pH were $50^{\circ}C$ and pH 8.0. The ranges of its stability to the pH and temperature were 7.0 to 9.0 and under $40^{\circ}C$, respectively. The enzyme was highly stable in the presence of the surfactants like Triton X-100 (0.1%), showing a 2-fold increase in its proteolytic activity. However, the enzyme was slightly inhibited by the chelating agent EDTA (1 mM). The enzyme has a maximum activity at $50^{\circ}C$ and the activity can be increased by surfactants such as Triton X-100 and Tween 80.

• Journal of Microbiology and Biotechnology
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• 제13권2호
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• pp.230-235
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• 2003

A 1.25 kb DNA fragment including the lscR gene, which encodes a levansucrase of Rahnella aquatilis ATCC 15552, was subcloned into a high-expression vector, pET-29b, and the recombinant enzyme was overexpressed in Escherichia coli. Most of the levansucrase activity was detected in the cytoplasmic fraction after induction with isopropyl ${\beta}-D-thiogalactoside$. The recombinant enzyme with a tag of six histidine residues at the C-terminus was purified 146-fold by affinity and gel-filtration chromatographies. The molecular mass of the purified LscR was approx. 49 kDa as determined by SDS-PAGE. The optimum pH and temperature of this enzyme for levan formation was pH 6.0 and $30^{\circ}C$, respectively. The optimum substrate concentration for levan formation was 300 mM sucrose. Levan formation was increased by the increase of the enzyme concentrations. Maxium yield of levan formation at optimum substrate concentration, pH, and temperature after 24 h of reaction was approximately 80%.

• Journal of Microbiology and Biotechnology
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• 제20권12호
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• pp.1681-1688
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• 2010

A screening for cellobiohydrolase (CBH) activity was performed and Fomitopsis pinicola KMJ812 was selected for further characterization as it produced a high level of CBH activity. An extracellular CBH was purified to homogeneity by sequential chromatography of F. pinicola culture supernatants. The molecular mass of the F. pinicola CBH was determined to be 64 kDa by SDS-PAGE and by size-exclusion chromatography, indicating that the enzyme is a monomer. The F. pinicola CBH showed a $t_{1/2}$ value of 42 h at $70^{\circ}C$ and catalytic efficiency of $15.8mM^{-1}s^{-1}(k_{cat}/K_m)$ for p-nitrophenyl-${\beta}$-D-cellobioside, one of the highest levels seen for CBH-producing microorganisms. Its internal amino acid sequences showed a significant homology with hydrolases from glycoside hydrolase family 7. Although CBHs have been purified and characterized from other sources, the F. pinicola CBH is distinguished from other CBHs by its high catalytic efficiency and thermostability.

• The Plant Pathology Journal
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• 제32권5호
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• pp.452-459
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• 2016

The genomic region of Grapevine fanleaf virus (GFLV) encoding the movement protein (MP) was cloned into pET21a and transformed into Escherichia coli strain BL21 (DE3) to express the protein. Induction was made with a wide range of isopropyl-${\beta}$-D-thiogalactopyranoside (IPTG) concentrations (1, 1.5, and 2 mM) each for duration of 4, 6, or 16 h. However, the highest expression level was achieved with 1 mM IPTG for 4 h. Identity of the expressed protein was confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting. The expressed 41 kDa protein was purified under denaturing condition by affinity chromatography, reconfirmed by Western blotting and plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA) before being used as a recombinant antigen to raise polyclonal antibodies in rabbits. Purified anti-GFLV MP immunoglobulines (IgGs) and conjugated IgGs detected the expressed MP and GFLV virions in infected grapevines when used in PTA-ELISA, double antibody sandwich-ELISA, and Western blotting. This is the first report on the production of anti-GFLV MP polyclonal antibodies and application for the virus detection.

• Reproductive and Developmental Biology
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• 제29권3호
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• pp.187-191
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• 2005

본 연구는 한우 난포란이 체외성숙된 환경의 변화를 단백질 측면으로부터 검토하기 위하여 체외성숙 배지와 세포질내 단백질 변화와 종류를 검토하였다. 그 결과 배지 내의 단백질 발현량은 배양 4.5시간째까지 감소하였고, 배양 13.5시간째까지는 변화가 없었다. 그러나 배양 $13.5\~18$시간 사이에 증가한 후 배양 18시간 이후에 다시 감소하는 경향이었다. 세포질내의 단백질 발현량은 배양 4.5시간째까지 증가하였고 배양 9시간째까지 급격히 감소하였다. 배양 9시간째부터 18시간째 까지는 단백질 발현량이 유사한 경향이었으나 배양 18시간째부터 24시간째까지 다시 증가하였다. 한편 체외성숙한 배지와 세포질을 2차원 전기영동하여 각각 298개 및 35개의 단백질 spot을 확인하였고, 그 중 배지에서는 28개, 세포질에서는 5개의 spot이 유의적인 변화를 확인하였다. 이들 spot 대한MALDI-TOP분석으로 배지와 세포질에서 각각 8개 및 1개의 단백질을 동정하였다. 그 종류는 aldose reductase, alpha enolase, apolipoprotein A-1 precursor, 43M1a collectin precursor, heat shock 27kDa protein, plasminogen activator inhibitor-1 precursor, thrombospondin 1 transitional endoplasmic reticulum ATPase 및 $\beta$-tubulin이었다.

• Journal of Microbiology and Biotechnology
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• 제10권1호
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• pp.56-62
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• 2000

The hpa gene genetically linked to the ctxa2b gene was cloned into the pTED expression vector, and the constructed pTEDhpa/ctxa2b was transformed into Excherichia coli. The fusion protein, the adhesin fused to the cholera toxin subunit A2B (CTXA2B) subunit, was expressed to high levels as inclusion bodies in E. coli. The expressed protein was partially purified by washing the inclusion bodies with working solution containing 8M Urea and 0.1M DTT. Refolding of denatured fusion protein was carried out in the presence of glutathione redox buffer. The refolded fusion protein was purified by size exclusion chromatography. The expressed fusion protein was verified by SDS-PAGE, western blotting with antibodies to both antigenic components of adhesin and cholera toxin subunit B (CTXB), and its N-terminal amino acid sequence was analyzed. The orderly assembled fusion protein was confirmed by modified Gm1-ganglioside ELISA with Abs to adhesin. The results indicate that the purified fusion protein is an Adhesin/CTXA2B protein containing the H. pylori adhesin and $G_{m1}4-ganglioside binding activity of CTXB and the expressed fusion protein in E. coli could be easily purified by the refolding process, Its molecular weight was 168kDa as estimated by size exclusion chromatography. The Adhesin/CTXA2B protein may be used as a candidate antigen for oral immunization against H. pylori.

• 한국작물학회지
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• 제43권4호
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• pp.259-263
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• 1998

High molecular weight glutenin (HMW-Glu) subunit compositions of 73 Korean wheat cultivars and experimental lines were evaluated by using one dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This method is suitable for obtaining a good resolution of 1Dx2 and 1Ax2$^*$ without adverse effects on separation of other HMW-Glu subunits. Korean wheats examined in this study could be divided into 15 different groups on the basis of HMW-Glu subunit compositions. From the wheat lines tested, it was identified that there were three alleles at the Glu-Al, five at the Glu-Bl and three at the Glu-D1 loci. The null allele of the Glu-Al was occurred in high frequency (79.4%), while low frequencies for 1Ax1 (12.3%) and 1Ax2$^*$(8.2%) were found. High frequency (75.3%) of the subunit pairs of 1Bx7+1By8 at the Glu-Bl loci compared with other subunits was found. The frequencies of subunits 1Dx2. 2+1Dy12 and 1Dx2+1Dy12 from the Glu-D1 loci were 54. 8% and 37.0%, respectively. However, a few Korean wheat lines (8.2%) carried 1Dx5 + 1Dy10 subunit pair which are responsible for good breadmaking quality. The information of HMW-Glu subunit compositions provide a useful tool to characterize wheat lines, and can be directly used in selection of breeding lines of different end-use properties.

• 한국식품과학회지
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• 제36권1호
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• pp.152-157
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• 2004

효소에 의한 단백질 분해로 우리 밀의 저 알레르겐화를 시도하기 위한 기초 연구로서 금강밀에서 gluten fraction을 추출하여 동물 실험을 통하여 경구 섭취된 gluten에 대한 항체 생성을 유도하였다. Gluten fraction은 UTH buffer로 추출하여 10-50kDa 사이의 분자량에서 여러 밴드가 확인된 분획을 얻었으며 약 25 kDa, 34 kDa 그리고 45 kDa 부근의 밴드가 주요하게 나타났다. 그리고, 경구 투여로 유도된 gluten에 대한 항체는 ELISA와 western blot의 항원-항체 반응을 실시하여 anti-gluten lgG, IgE를 확인하였다. 또한 경구 투여의 투여 시간 경과에 따라 항체가는 상승하였다. 추출된 gluten fraction은 bromelain, papain, ficin 그리고 b.p. protease 각 효소의 최적 pH와 온도에서 가수 분해하여 gluten fraction의 알레르겐 저감화를 시도하였다. 효소 분해 후 gluten fraction의 저분자화 정도는 anti-gluten 하체에 대한 ELISA를 실시하여 검토하였다. 그 결과 bromelain, papain, ficin의 경우보다 b.p. protease을 처리한 경우가 ELISA value를 크게 저하시켰다. 이것은 gluten fraction이 b.p protease 효소 가수분해에 의해 생성된 항체가 인식하는 amino acid 서열 (epitope)이 분해되었음을 나타낸 것이다. 마지막으로 b.p. protease의 분해능력을 1mg, 2mg, 3mg/10mL로 첨가량을 달리하여 검토한 결과 첨가량에 따른 변화는 거의 나타나지 않았다. 따라서 금강밀의 gluten fraction 10mL에 b.p protease 1mg으로 4시간 처리하여 단백질을 분해하여 우리밀의 항원성 저하 가능성이 탐색되었다.