• Title/Summary/Keyword: Efficient Purification

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Expression, Purification, and Biological Characterization of The Amino-Terminal Fragment of Urokinase in Pichia pastoris

  • Li, Jianping;Lin, Yuli;Zhuang, Hongqin;Hua, Zi-Chun
    • Journal of Microbiology and Biotechnology
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    • v.23 no.9
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    • pp.1197-1205
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    • 2013
  • Urokinase (uPA) and its receptor (uPAR) play an important role in tumor growth and metastasis. Targeting the excessive activation of this system as well as the proliferation of the tumor vascular endothelial cell would be expected to prevent tumor neovasculature and halt the tumor development. In this regard, the amino-terminal fragment (ATF) of urokinase has been confirmed as effective to inhibit the proliferation, migration, and invasiveness of cancer cells via interrupting the interaction of uPA and uPAR. Previous studies indicated that ATF expressed in Escherichia coli was mainly contained in inclusion bodies and also lacked posttranslational modifications. In this study, the biologically active and soluble ATF was cloned and expressed in Pichia pastoris. The recombinant protein was purified to be homogenous and confirmed to be biologically active. The yield of the active ATF was about 30 mg/l of the P. pastoris culture medium. The recombinant ATF (rATF) could efficiently inhibit angiogenesis, endothelial cell migration, and tumor cell invasion in vitro. Furthermore, it could inhibit in vivo xenograft tumor growth and prolong the survival of tumor-bearing mice significantly by competing with uPA for binding to cell surfaces. Therefore, P. pastoris is a highly efficient and cost-effective expression system for large-scale production of biologically active rATFs for potential therapeutic application.

Improvement of a Sulfolobus-E. coli Shuttle Vector for Heterologous Gene Expression in Sulfolobus acidocaldarius

  • Hwang, Sungmin;Choi, Kyoung-Hwa;Yoon, Naeun;Cha, Jaeho
    • Journal of Microbiology and Biotechnology
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    • v.25 no.2
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    • pp.196-205
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    • 2015
  • A Sulfolobus-E. coli shuttle vector for an efficient expression of the target gene in S. acidocaldarius strain was constructed. The plasmid-based vector pSM21 and its derivative pSM21N were generated based on the pUC18 and Sulfolobus cryptic plasmid pRN1. They carried the S. solfataricus P2 pyrEF gene for the selection marker, a multiple cloning site (MCS) with C-terminal histidine tag, and a constitutive promoter of the S. acidocaldarius gdhA gene for strong expression of the target gene, as well as the pBR322 origin and ampicillin-resistant gene for E. coli propagation. The advantage of pSM21 over other Sulfolobus shuttle vectors is that it contains a MCS and a histidine tag for the simple and easy cloning of a target gene as well as one-step purification by histidine affinity chromatography. For successful expression of the foreign genes, two genes from archaeal origins (PH0193 and Ta0298) were cloned into pSM21N and the functional expression was examined by enzyme activity assay. The recombinant PH0193 was successfully expressed under the control of the gdhA promoter and purified from the cultures by His-tag affinity chromatography. The yield was approximately 1 mg of protein per liter of cultures. The enzyme activity measurements of PH0913 and Ta0298 revealed that both proteins were expressed as an active form in S. acidocaldarius. These results indicate that the pSM21N shuttle vector can be used for the functional expression of foreign archaeal genes that form insoluble aggregates in the E. coli system.

Separation of Lysozyme from Chicken Egg White by Ultrafiltration (1) - Dead-end Filtration of Egg White Solution - (한외여과에 의한 계란 난백으로부터 라이소자임의 분리 (1) - 난백용액의 전량여과 -)

  • Park, Hye-Ri;Youm, Kyung-Ho
    • Membrane Journal
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    • v.26 no.4
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    • pp.266-272
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    • 2016
  • The separation and purification of lysozyme enzyme from chicken egg white (CEW) solution was studied using the dead-end filtration. The optimum operation conditions of the dead-end filtration reveal that the maximum value of permselectivity of lysozyme to the other proteins of ovalbumin and conalbumin in the CEW solution was tested with change of molecular weight cut-off (MWCO) of ultrafitration membrane and pH of the CEW solution. The optimum operation conditions for the efficient permeable separation of lysozyme from the CEW solution are that the membrane MWCO is 30 kD and the pH of CEW solution is 11. At this optimum operation conditions, the maximum value of permselectivity of lysozyme to total proteins in CEW solution is about 83.

A Study on Application and Verification of Heavy Water Treatment Effects Using Plant Cultivation (Vegetation) on Floating Island (식생섬에 의한 중수처리 효과 검증과 적용에 관한 연구)

  • Kwon, Dong Min;Kwon, Soon Hyo;Koo, Bon Hak
    • Journal of the Korean Society of Environmental Restoration Technology
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    • v.15 no.6
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    • pp.53-59
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    • 2012
  • This this study was conducted with the aim of doing experiment on the effect of water purification by using an artificially built plant island, which is one of the eco-techniques, and aquatic plants as a plan for the reuse of water for obtaining water resources, thereby analyzing the removed quantity, and applying the experimental results to the reuse of water. As a result of doing experiments, this study obtained a good measured value of BOD (biochemical oxygen demand) 4.7mg/L, and COD (chemical oxygen demand) 7.2mg/L below the heavy water standard of BOD 10mg/L and COD 20mg/L, respectively. The chromaticity showed 89.2% removal efficiency, but final treated wastewater was found to show chromaticity 58 degrees exceeding chromaticity 20 degrees which are the water quality standard of the reuse of water. The results revealed that T-N produced 27% removal efficiency on an average while T-P produced 38% removal efficiency on an average, showing that the removal effect of N & P wasn't big. According to the currently enforced "Water Quality Standard of Heavy Water by Use", the use of water for sprinkling and landscaping was found to be available. Accordingly, this study suggested a nature-friendly, economically-efficient, and eco-technological water treatment technique which will make it possible to overcome the limit of the existing physio-chemical water treatment technology, reduce the costs for maintenance and facilities, and also reduce the limit of space restraint for installation of facilities.

Visible Light-based Photocatalytic Degradation by Transition Metal Oxide (전이 금속 산화물을 이용한 가시광선 기반 광촉매 분해)

  • Lee, Soomin;Park, Yeji;Lee, Jae Hun;Patel, Rajkumar
    • Membrane Journal
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    • v.29 no.6
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    • pp.299-307
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    • 2019
  • Photocatalysis is an environment friendly technique for degrading organic dyes in water. Tungsten oxide is becoming an active area of research in photocatalysis nanomaterials for having a smaller bandgap than the previously favored titanium dioxide. Synthesis of hierarchical structures, doping platinum (Pt), coupling with nanocomposites or other semiconductors are investigated as valid methods of improving the photocatalytic degradation efficiency. These impact the reaction by creating a redshift in the wavelength of light used, effecting charge transfer, and the formation/recombination of electron-hole pairs. Each of the methods mentioned above are investigated in terms of synthesis and photocatalytic efficiency, with the simplest being modification on the morphology of tungsten oxide, since it does not need synthesis of other materials, and the most efficient in photocatalytic degradation being complex coupling of metal oxides and carbon composites. The photocatalysis technology can be incorporated with water purification membrane by modularization process and applied to advanced water treatment system.

Spectrophotometric Determination of Traces of Boron in Semiconductor-grade Trichlorosilane (반도체급 삼염화실란중의 극미량 붕소의 분광 광도법적 측정)

  • Dong Kwon Kim;Hee Young Kim
    • Journal of the Korean Chemical Society
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    • v.35 no.5
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    • pp.534-538
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    • 1991
  • A procedure for spectrophotometric determination of traces of boron in high-purity trichlorosilane (TCS) is proposed utilizing an adsorptive separation. NaCl is chosen as an Lewis base adsorbent which forms a complex with boron compounds in TCS, and is well dissolved in sulfuric acid-quinalizarin color-forming agent without causing an interference in colorimetric measurements. The proposed adsorptive separation method is free from the formation of silica gel and gas bubbles during the analysis of TCS. The method reveals that the boron concentration in a semiconductor grade TCS is 6.1 ${\mu}$g/l within the standard deviation of ${\pm}$20%. On the other hand, the boron concentration of the purified TCS which is separated from NaCl-boron compounds complex is reduced to 0.2 ${\mu}$g/l, showing the efficient applicability of NaCl to the adsorptive separation. The effectiveness of NaCl for the removal of boron in TCS purification is also described in comparison with other well-known adsorbents.

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Metabolic Profiling and Biological Activities of Bioactive Compounds Produced by Pseudomonas sp. Strain ICTB-745 Isolated from Ladakh, India

  • Kama, Ahmed;Shaik, Anver Basha;Kumar, C. Ganesh;Mongolla, Poornima;Rani, P. Usha;Krishna, K.V.S. Rama;Mamidyala, Suman Kumar;Joseph, Joveeta
    • Journal of Microbiology and Biotechnology
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    • v.22 no.1
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    • pp.69-79
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    • 2012
  • In an ongoing survey of the bioactive potential of microorganisms from Ladakh, India, the culture medium of a bacterial strain of a new Pseudomonas sp., strain ICTB-745, isolated from an alkaline soil sample collected from Leh, Ladakh, India, was found to contain metabolites that exhibited broad-spectrum antimicrobial and biosurfactant activities. Bioactivity-guided purification resulted in the isolation of four bioactive compounds. Their chemical structures were elucidated by $^1H$ and $^{13}C$ NMR, 2D-NMR (HMBC, HSQC, $^1H$,$^1H$-COSY, and DEPT-135), FT-IR, and mass spectroscopic methods, and were identified as 1-hydroxyphenazine, phenazine-1-carboxylic acid (PCA), rhamnolipid-1 (RL-1), and rhamnolipid-2 (RL-2). These metabolites exhibited various biological activities like antimicrobial and efficient cytotoxic potencies against different human tumor cell lines such as HeLa, HepG2, A549, and MDA MB 231. RL-1 and RL-2 exhibited a dose-dependent antifeedant activity against Spodoptera litura, producing about 82.06% and 73.66% antifeedant activity, whereas PCA showed a moderate antifeedant activity (63.67%) at 60 ${\mu}g/cm^2$ area of castor leaf. Furthermore, PCA, RL-1, and RL-2 exhibited about 65%, 52%, and 47% mortality, respectively, against Rhyzopertha dominica at 20 ${\mu}g/ml$. This is the first report of rhamnolipids as antifeedant metabolites against Spodoptera litura and as insecticidal metabolites against Rhyzopertha dominica. The metabolites from Pseudomonas sp. strain ICTB-745 have interesting potential for use as a biopesticide in pest control programs.

The Methodology for Investigation on Seabed Litters and The Distribution of Seabed Litters in Ports around the Korean Coastline (수중침적 폐기물 실태조사 및 국내 연안의 항 내 수중침적 폐기물 분포에 관한 연구)

  • Kang W.S.
    • Journal of the Korean Society for Marine Environment & Energy
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    • v.4 no.3
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    • pp.74-80
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    • 2001
  • Due to floating marine debris, seabed litters, dead shellfishes and polluted sediments, etc. , which are mainly caused by fishery activities in a large scale around the coastline, expansion of industrialized areas from economic development and drastic increase of free time by the improved standard of living, Korean coast is believed to be cast beyond the self purification of marine ecosystem. Seabed litters, if not remedied in a timely manner, will eventually lead to the overall disorder of benthic ecosystem. Thus, in order to prevent marine ecosystem from being thrown into confusion by seabed litters and to restore ocean environments, it is reasonable to investigate the current status of the seabed litters by carrying out an extensive examination on them and to equip ourselves for marine debris with an systemized tool that provides supports to the building process of an efficient methodology of litters disposal through managing related data appropriately. As an primary step to this goal, a systematic method of investigation is presented in this paper. With this methodology, the present state of the seabed titters is properly understood for the specified list of ports.

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Efficient bio-gas desulfurization purification technology development Using ion-exchange fibers (이온교환섬유를 이용한 바이오가스 고효율 탈황정제기술 개발)

  • Tak, Bong-Yeol;Tak, Bong-Sik;Min, Gil-Ho;Lee, Sang-Min;Lee, Won-Gu;Lee, So-A
    • 한국신재생에너지학회:학술대회논문집
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    • 2011.11a
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    • pp.116-116
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    • 2011
  • 바이오 가스 플랜트의 혐기소화 공정에서 발생하는 바이오 가스는 중 유해가스인 황하수소($H_2S$)는 부식성 가스로 수천 PPM농도를 함유하여, 발전기나 가스보일러로 이용하는 경우에는 $H_2S$를 제거하는 탈황공정이 반드시 필요하다. 탈황방식에는 산화철 탈황(건식 탈황)과 생물 탈황이 현재 많이 사용되고 있어나 산화철 탈황은 산화철 pellet이 유화철에 변화하면 탈황능력이 저하되어 pellet을 교환해야 하며 많은 비용이 발생한다. 생물 탈황 방식은 유황산화세균의 서식활동조건(온도, 반응시간, 산소량)확보가 반드시 필요하여 높은 운전기술을 필요로 한다. 본 연구에서는 바이오가스 전처리 기술 중 활성탄 또는 약액을 이용한 기존의 탈황정제방식보다 흡착성능이 뛰어난 이온교환섬유를 이용하여, 황화수소($H_2S$)를 95% 이상 제거할 수 있는 고효율 섬유상 이온촉매 악취제거 시스템 개발을 수행하였다. 이온교환섬유는 방사선 조사를 이용하여 부직포에 라디칼을 인위적으로 형성시켜(그라프트 중합) 양이온 또는 양이온을 교환할 수 있도록 제조된 섬유상의 흡착제로, 이온교환 섬유의 화학적 이온교환과 물리적 흡착 및 탈착반응이 동시에 발생되고, 활성탄/실리카켈 보다 흡착능력이 2~4배 높다. 또한 이온섬유의 재생기능을 이용하여 장기적 다양한 악취($H_2S$, $NH_3$, 아민계, 메르갑탄류, 알데히드 등) 및 유해가스(VOCs, NOx, SOx) 등을 95% 이상 제거할 수 있다.

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Reduction of perchlorate in aqueous solution using zero valence iron stabilized with alginate bead (알지네이트 비드를 이용하여 안정화한 0가 철의 수용액 상에서의 과염소산 이온의 환원 분해 특성)

  • Joo, Tae-Kyeong;Lee, Jong-Chol;Paeng, Ki-Jung
    • Analytical Science and Technology
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    • v.23 no.6
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    • pp.560-565
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    • 2010
  • Perchlorate ion ($ClO_4^-$) has been widely used as oxidizing agent in military weapon system such as rocket and missile fuel propellant. So it has been challenging to remove the pollutant of perchlorate ion. nanoscale zero valence iron (nZVI) particles are widely employing reduction catalyst for decomposition of perchlorate ion. nZVI particles has increasingly been utilized in groundwater purification and waste water treatment. But it have strong tendency of aggregation, rapid sedimentation and limited mobility. In this study, we focused on reduction of perchlorate ion using nZVI particles immobilized in alginate polymer bead for stabilization. The stabilized nZVI particles displayed much greater surface area, and much faster reaction rates of reduction of perchlorate ion. In this study, an efficient way to immobilize nZVI particles in a support material, alginate bead, was developed by using $Ca^{2+}$ as the cross-linking cations. The efficiency and reusability of the immobilized Fe-alginate beads on the reduction of perchlorate was tested at various temperature conditions.