• Title/Summary/Keyword: size exclusion

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NMR and Fluorescence Studies of DNA Binding Domain of INI1/hSNF5

  • Lee, Dongju;Moon, Sunjin;Yun, Jihye;Kim, Eunhee;Cheong, Chaejoon;Lee, Weontae
    • Bulletin of the Korean Chemical Society
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    • v.35 no.9
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    • pp.2753-2757
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    • 2014
  • INtegrase Interactor 1 protein (INI1/hSNF5) or BRG1-associated factor 47 (BAF47) is a SWI/SNF-related matrix associated actin dependent regulator of chromatin subfamily B member. DNA binding domain of INI1/hSNF5 is cloned into E.coli expression vectors, pET32a and purified as a monomer using size exclusion chromatography. NMR data show that $INI1^{DBD}$ has folded state with high population of ${\alpha}$-helices. By fluorescence-quenching experiments, binding affinities between $INI1^{DBD}$ and two double stranded DNA fragments were determined as $29.9{\pm}2.6{\mu}M$ (GAL4_1) and $258.7{\pm}5.8$ (GAL4_2) ${\mu}M$, respectively. Our data revealed that DNA binding domain of INI1/hSNF5 binds to transcriptional DNA sequences, and it could play an important role as a transcriptional regulator.

Analysis of the Structure and Stability of Erythropoietin by pH and Temperature Changes using Various LC/MS

  • Chang, Seong-Hun;Kim, Hyun-Jung;Kim, Chan-Wha
    • Bulletin of the Korean Chemical Society
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    • v.34 no.9
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    • pp.2663-2670
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    • 2013
  • The purpose of stability testing is to provide evidence about how the quality of a drug varies with time under the influence of a variety of environmental factors. In this study, erythropoietin (EPO) was analyzed under different pH (pH 3 and pH 9) and temperature ($25^{\circ}C$ and $40^{\circ}C$) conditions according to current Good Manufacturing Practice (cGMP) and International Conference on Harmonisation (ICH) guidelines. The molecular weight difference between intact EPO and deglycosylated EPO was determined by SDS-PAGE, and aggregated forms of EPO under thermal stress and high-pH conditions were investigated by size exclusion chromatography. High pH and high temperature induced increases in dimer and high molecular weight aggregate forms of EPO. UPLC-ESI-TOF-MS was applied to analyze the changed modification sites on EPO. Further, normal-phase high-performance liquid chromatography was performed to identify proposed glycan structures and high pH anion exchange chromatography was carried out to investigate any change in carbohydrate composition. The results demonstrated that there were no changes in modification sites or the glycan structure under severe conditions; however, the number of dimers and aggregates increased at $40^{\circ}C$ and pH 9, respectively.

Removal Characteristics of Endocrine Disrupting Compounds (EDCs), Pharmaceutically Active Compounds (PhACs) and Personal Care Products (PCPs) by NF Membrane (NF막을 이용한 EDCs, PhACs, PCPs 물질의 제거 특성 평가)

  • Jang, Hyuewon;Park, Chanhyuk;Hong, Seungkwan;Yoon, Yeomin;Jung, Jin-Young;Chung, Yun-Chul
    • Journal of Korean Society of Water and Wastewater
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    • v.21 no.3
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    • pp.349-357
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    • 2007
  • Reports of endocrine disrupting compounds (EDCs), pharmaceutically active compounds (PhACs), and personal care products (PCPs) have raised substantial concern in important potable drinking water quality issues. Our study investigates the removal of EDCs, PhACs, and PCPs of 10 compounds having different physico-chemical properties (e.g., molecular weight, and octanol-water partition coefficient ($K_{OW}$)) by nanofiltration (NF) membranes. The rejection of micropollutants by NF membranes ranged from 93.9% to 99.9% depending on solute characteristics. A batch adsorption experiments indicated that adsorption is an important mechanism for transport/removal of relatively hydrophobic compounds, and is related to the octanol-water partition coefficient values. The transport phenomenon associated with adsorption may also depend on solution water chemistry such as pH and ionic strength influencing the pKa value of compounds. In addition, it was visually seen that the retention was somewhat higher for the larger compounds based on their molecular weight. These results suggest that the NF membrane retains many organic compounds due to both hydrophobic adsorption and size exclusion mechanisms.

Characteristics of pre-extracted hemicelluloses from Korean mixed wood by hot water and alkali solution and its effect on handsheet properties (열수 및 알칼리 용액을 이용하여 국산 목재 칩으로부터 선추출한 헤미셀룰로오스의 특성과 이에 따른 수초지 물성 변화)

  • Seo, Dong-Il;Lee, Sang-Hoon;Sim, Kyu-Jeong;Lee, Hak-Lae;Youn, Hye-Jung
    • Journal of Korea Technical Association of The Pulp and Paper Industry
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    • v.43 no.5
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    • pp.60-67
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    • 2011
  • Hemicelluloses pre-extracted from Korean mixed wood chip were investigated as a wet-end additive. Hemicelluloses dissolved in hot water and alkali solution were isolated by ethyl alcohol precipitation from pre-extractives. They showed molecular weight of 9,000 ~ 27,000 g/mol as revealed by size exclusion chromatography. The reduction of molecular weight through hot water extraction was caused by autohydrolysis. Chemical composition of the hemicelluloses were analyzed with high-performance liquid chromatography and UV-Vis spectroscopy. As the surface charge of isolated hemicelluloses were negative, the adsorption of hemicelluloses onto softwood unbleached kraft pulp fiber was promoted by poly-DADMAC. The physical properties of handsheets increased as the molecular weight of hemicellulose increased. On the other hands, the optical property decreased with hemicellulose adsorption.

REVIEW OF METHODS FOR PROCESSING ALLOGRAFTS FOR ALVEOLAR BONE RECONSTRUCTION (치조골 재생술에 사용되는 동종골 처리방법에 대한 고찰)

  • Lee, Eun-Young;Kim, Kyoung-Won;Um, In-Woong
    • Maxillofacial Plastic and Reconstructive Surgery
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    • v.29 no.4
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    • pp.366-371
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    • 2007
  • Evaluation of the methods of processing allogenic bone must be considered in order to make an effective choice of graft materials in oral surgery. Allograft materials processed by the tissue banking industry have varying capacities of bone reconstruction. The biological function of processed bone can be affected by many factors, like particle size, processing parameters, and inclusion or exclusion of mineral and moisture. For example, freeze drying step offers a safe and economical means for packaging, shipping, storage, and preservation of homologous bone. Demineralization of cortical bone using hydrochloric acid can produce a uniform demineralized surface with a capacity for osteoinduction. The objectives of this review were to evaluate the processing methods for allogenic bone and to characterize processed materials for grafting. It is important to understand the biological, biomechanical healing of different types of allografts to make the right choice for allogenic bone on each clinical application and to achieve a successful outcome for alveolar bone reconstruction in oral surgery.

Isolation and Characterization of Chondroitin Sulfates from the Byproducts of Marine Organisms

  • Im, A-Rang;Sim, Joon-Soo;Park, You-Mie;Hahn, Bum-Soo;Toida, Toshihiko;Kim, Yeong-Shik
    • Food Science and Biotechnology
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    • v.18 no.4
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    • pp.872-877
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    • 2009
  • By-products of marine organisms including salmon, skate, flatfish, and yellow goosefish were investigated to search for new source of chondroitin sulfate (CS). Agarose gel electrophoresis with chondroitinase depolymerization showed that purified chondroitin sulfate did not contain any other glycosaminoglycans. 1H-nuclear magnetic resonance (NMR) spectra were acquired to confirm the structure and purity. The average molecular weight ranging from 22 to 64 kDa was determined by high performance size exclusion chromatography. Disaccharide compositions and purities were determined by strong anion exchange-high performance liquid chromatography (SAX-HPLC) after chondroitinase ABC depolymerization. SAX-HPLC data exhibited that the purity was from $81.7{\pm}1.3$ to $114.2{\pm}2.5%$ and the yield was from 1.3 to 12.5%. All analytical results indicate that salmon cartilage, skate cartilage, and yellow goosefish bone could be promising sources of CS to substitute shark cartilage CS in commercial neutraceuticals.

Rapid Separation of Cellular Cyclosophoraoses Produced by Rhizobium Species

  • Seo, Dong-Hyuk;Lee, Sang-Hoo;Park, Hey-Lin;Kwon, Tae-Jong;Jung, Seun-Ho
    • Journal of Microbiology and Biotechnology
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    • v.12 no.3
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    • pp.522-525
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    • 2002
  • A very rapid and efficient separation technique for cellular rhizobial cyclosophoraoses was developed based on fractional precipitation and partition chromatography. Cyclosophoraoses are known to function in the osmotic regulation and root nodule formation of legumes during the nitrogen fixation process. Cyclosophoraoses are produced as unbranched cyclic (1longrightarrow12)-${\beta}$-D-glucans in Agrobacterium or Rhizobium species. Recent research has shown that cyclosophoraoses can form inclusion complexation with various unstable or insoluble guest chemicals, thereby implying great potential for industrial application. Typical separation of pure cellular cyclosophoraoses has been so far carried out by several time-consuming steps, including size exclusion, anion exchange, and desalting liquid chromatographies, with a relatively poor recovery. However, the proposed method demonstrated that the successive application of fractional ethanol precipitation and one step of silica gel-based flash column chromatography was enough to simultaneously purify neutral or anionic forms of cyclosophoraoses. This novel technique is very rapid and provides a high recovery.

Design and Expression of Recombinant Antihypertensive Peptide Multimer Gene in Escherichia coli BL21

  • Rao, Shengqi;Su, Yujie;Li, Junhua;Xu, Zhenzhen;Yang, Yanjun
    • Journal of Microbiology and Biotechnology
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    • v.19 no.12
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    • pp.1620-1627
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    • 2009
  • The design and expression of an antihypertensive peptide multimer (AHPM), a common precursor of 11 kinds of antihypertensive peptides (AHPs) tandemly linked up according to the restriction sites of gastrointestinal proteases, were explored. The DNA fragment encoding the AHPM was chemically synthesized and cloned into expression vector pGEX-3X. After an optimum induction with IPTG, the recombinant AHPM fused with glutathione S-transferase (GST-AHPM) was expressed mostly as inclusion body in Escherichia coli BL21 and reached the maximal production, 35% of total intracellular protein. The inclusion body was washed, dissolved, and purified by cation-exchange chromatography under denaturing conditions, followed by refolding together with size-exclusion chromatography and gradual dialysis. The resulting yield of the soluble GSTAHPM (34 kDa) with a purity of 95% reached 399 mg/l culture. The release of high active fragments from the AHPM was confirmed by the simulated gastrointestinal digestion. The results suggest that the design strategy and production method of the AHPM will be useful to obtain a large quantity of recombinant AHPs at a low cost.

Characterization of β-glucosidase from Brown Rot Fungus, Laetiporus sulphureus

  • Lee, Jae-Won;Park, Jun-Yeong;Gwak, Ki-Seob;Koo, Bon-Wook;Choi, In-Gyu
    • Journal of the Korean Wood Science and Technology
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    • v.35 no.5
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    • pp.100-108
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    • 2007
  • $\beta$-Glucosidase from Laetiporus sulphureus among the enzymes related to lignocellulosic biomass degradation to sugars for using alternative bioethanol production was characterized. The highest activity of $\beta$-glucosidase was obtained on cellobiose at shaking culture. For the characterization and purification of $\beta$-glucosidase culture solution was concentrated and then purified by FPLC using ion exchange and size exclusion column. According to the results of SDS-PAGE, native PAGE and microfluidic system of purified enzyme, protein band was observed at about 132 kDa. Optimal pH and temperature of purified $\beta$-glucosi-dase were 5.0 and $60^{\circ}C$, respectively. In the kinetic properties of $\beta$-glucosidase on various substrates such as sophorose, gentiobiose and cellobiose, $K_m$ was 0.81, 1.07 and 1.70 mM, respectively.

Anti-cell Adhesion Effect of PLM-f74 with U937 Cell from Hallophilic Enterobacteria and Identification of Strain

  • Lim, Jong-Kwon;Seo, Hyo-Jin;Shin, Jin-Hyuk;Lee, Se-Young;Kim, Min-Yong;Kim, Jong-Deog
    • 한국생물공학회:학술대회논문집
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    • 2005.10a
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    • pp.406-411
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    • 2005
  • Fermented materials with enterobacteria isolated from fusiform fish, have strong anti-angiogenesis effect and anti-cell adhesion effect. PLM-f74 got from 74th fraction of size exclusion chromatography from fermented material, showed strong anti-cell adhesion effect between HUVECs and U937 monocytic cell. Adhesion of U937 cell to HUVEC stimulated with IL-1b was clearly inhibited by PLM-f74 in a dose-dependent manner by 12.1, 21.2, 50.9, and 78.2%, when U937 cells treated with each of the PLM-f74 and stimulated with PMA (100 mg/L) was added onto untreated and unstimulated HUVECs, adhesion was observed by 15.8, 31.9, 70.8, and 102%, when both cell types were pretreated with PLM-f74, the adhesion was prominently decreased by 83.7, 99.2, 110, and 120.8%, with 0.74, 3.7, 7.4, and 18.5ug/mL of PLM-f74, respectively. PLM-f74, also, reduced IL-1-stimulated HUVEC expression of adhesion molecules, VCAM-1, ICAM-1, and E-selectin dose-dependently by ELISA method.

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