• Title/Summary/Keyword: molecular design

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Single-Chain Fv Fragment of Catalytic Antibody 4f4f with Glycosidase Activity: Design, Expression, and Purification

  • Jang, Chang-Hwan;Chung, Hyun-Ho;Yu, Jae-Hoon;Chang, Yung-Jin;Kim, Hyong-Bai;Paek, Se-Hwan;Shin, Dong-Hoon;Kim, Kyung-Hyun
    • Journal of Microbiology and Biotechnology
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    • v.9 no.3
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    • pp.376-380
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    • 1999
  • Constructs, encoding a single-chain variable fragment of a catalytic antibody 4f4f (scFv-4f4f) with glycosidase activity, were made by combining the coding sequences for the heavy and light chain variable domains with a sequence encoding a linker (GGGGS). Using three different plasmid systems, single-chain antibodies were expressed separately in Escherichia coli, demonstrating significant differences in the expression level and amounts in soluble form of the recombinant protein. The protein expression from pET3a-scFv-4f4f was up to 20% of the total soluble proteins and, more importantly, the proteins were mostly found in a soluble form. An SDS-PAGE analysis of the purified single-chain proteins, yielding higher than 5mg from a 1-1 culture, showed a single band corresponding to its molecular weight of 29,100. A preliminary study shows that the expressed scFv-4f4f is catalytically active. The catalytic parameters for the hydrolysis of p-nitrophenyl-$\beta$-D-glucopyranoside by scFv-4f4f are being investigated.

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Protein structure analysis : Pharmacophore study for new insecticide target AnCE using the substrate of ACE, HHL molecule (단백질의 구조연구 : ACE의 기질 HHL을 이용한 신규 살충제 표적 AnCE에 대한 약리단 연구)

  • Lee, Jung-Kyung;Kim, Kyeong-Yee
    • The Korean Journal of Pesticide Science
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    • v.9 no.3
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    • pp.191-198
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    • 2005
  • Hippuryl-L-histidyl-L-leucine (Hip-L-His-L-Leu, HHL) is the known substrate of ACE, which used often in inhibition kinetic study to design new inhibitor. Here we use HHL molecule as a template to predict pharmacophore which can interact with residues in active site of AnCE, new potential insecticide target protein. To explain physicochemical properties related to molecular geometry and conformational change in reaction field as well as electron density of atoms associated to pharmacophores, geometry optimization, NMR chemical shifts and natural population analysis were performed by ab initio and DFT method. Calculated NMR chemical shifts showed good agreement with the experimental ones and obtained electron densities were used for analyzing pharmacophores of corresponding atoms. Finally, we could extract aye pharmacophores related to hydrophobic aliphatic and aromatic site, hydrogen bonding donor and acceptor site and Zn binding site from the HHL molecule.

A Study on the Application of Soil Washing Technology for HOCs-Contaminated Soil Using Mixed Surfactants (소수성 유기오염물질로 오염된 토양에 대한 혼합 계면활성제를 이용한 토양세척기법의 적용성 연구)

  • Choi, Sang-Il;Lee, Jai-Young;Jang, Min
    • Journal of the Korean Society of Groundwater Environment
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    • v.4 no.2
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    • pp.103-108
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    • 1997
  • A series of batch tests were conducted to evaluate the design parameters for the application of soil washing techniques to the hydrophobic organic compounds (HOCs)-contaminated soil using mixed surfactants. Because the mixed surfactants form different structures of molecular aggregates from single surfactant, they were applied to improve the washing efficiency. Kinds of surfactants added, mixing ratio, and total concentration of mixed surfactants were evaluated. The uncontaminated soil was obtained from a country hill near Nock-Chun Station in Seoul. The portion of soil passing #4 (4.75 mm) sieve was used. The pH, organic contents and cation exchange capacity were 4.4, 1.6% and 4.08 meq/100 g, respectively The soil was artificially contaminated by n-dodecane. The 5% solution of OA-5 and OA-14 (1:1) showed 86% washing efficency. The 4% solution of SDS and OA-5 (1:1) showed 95% washing efficiency.

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Protective Effect of Radix Clematidis Extract on Streptozotocin-induced Diabetes (Streptozotocin 유도 당뇨병에 대한 위령선(威靈仙) 추출물의 방어 효과)

  • Ham, Kyung-Wan;Kim, Eun-Kyung;Song, Mi-Young;Kwon, Kang-Beom;Song, Je-Ho;Seo, Eun-A;Ryu, Do-Gon
    • Journal of Physiology & Pathology in Korean Medicine
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    • v.22 no.3
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    • pp.580-584
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    • 2008
  • In the present study, Radix clematidis extract (RCE) was evaluated to determine if it could protect pancreatic ${\beta}$ cells against multiple low dose streptozotocin (MLDS)-induced diabetes. Injection of mice with MLDS resulted in hyperglycemia and hypoinsulinemia, which was confirmed by immunohistochemical staining. However, the induction of diabetes by MLDS was completely prevented when mice were pre-administrated with RCE. Generation of oxidative stress is implicated in MLDS, a ${\beta}$ cell specific toxin-induced islet cell death. In this context, to elucidate the mechanisms of protective effects in RCE pre-administrated diabetic mice, we investigated the expression of heme oxygenase-1 (HO-1), which is one of the anti-oxidant enzymes. MLDS-induced HO-1 expressions were significantly reduced in MLDS-treated mice. However, the decrease of HO-1 by MLDS were protected by pretreatment of RCE. The molecular mechanism by which RCE inhibits diabetic conditions by MLDS appears to involve inhibition of HO-1 expression. Taken together, these results reveal the possible therapeutic value of RCE for the prevention of type 1 diabetes progression.

Protective Effect of Radix Trichosanthis Extracts on Cytotoxicity of Pancreatic ${\beta}-Cells$ by Cytokines (싸이토카인 유발 췌장 ${\beta}$세포 독성에 대한 천화분 추출물의 방어효과)

  • Song, Mi-Young;Kim, Eun-Kyung;Song, Je-Ho
    • Journal of Physiology & Pathology in Korean Medicine
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    • v.22 no.2
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    • pp.422-426
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    • 2008
  • In this study, the preventive effects of Radix Trichosanthis extracts (RTE) against cytokine-induced ${\beta}-cell$ death were assessed. Cytokines generated by immune cells infiltrating pancreatic islets are crucial mediators of ${\beta}-cell$ destruction in insulin-dependent diabetes mellitus. The treatment of RIN cells with $interleukin-1{\beta}$ ($IL-1{\beta}$) and $interferon-{\gamma}$ ($IFN-{\gamma}$) resulted in a reduction of cell viability. RTE protected $IL-1{\beta}$ and $IFN-{\gamma}$-mediated viability reduction in a concentration-dependent manner. Incubation with RTE also induced a significant suppression of $IL-1{\beta}$ and $IFN-{\gamma}$-induced inducible nitric oxide synthase (iNOS) protein expression. The molecular mechanism by which RTE inhibited iNOS protein expression appeared to involve the inhibition of $NF{-\kappa}B$ activation. The $IL-1{\beta}$ and $IFN-{\gamma}$-stimulated RIN cells showed increases in $NF{-\kappa}B$ binding activityand $I{\kappa}B{\alpha}$ degradation in cytosol compared to unstimulated cells. However, pretreatment with RTE inhibited cytokines-induced $I{\kappa}B{\alpha}$ degradation and $NF{-\kappa}B$ activation in RINm5F cells. Furthermore, the protective effects of RTE were verified via protection of impairment in glucose-stimulated insulin secretions in $IL-1{\beta}$ and $IFN-{\gamma}$-treated islets.

A Pilot-Scale Microfiltration/Ultrafiltration system for Drinking Water Treatment (상수처리를 위한 파일롯 규모의 정밀여과/한외여과 시스템)

  • Kim, Hanseung;Oh, Jeongik;Kim, Chunghwan
    • Journal of Korean Society of Water and Wastewater
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    • v.18 no.6
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    • pp.770-777
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    • 2004
  • Three pilot-scale membrane systems were operated using lake water as influent in this study. Microfiltration (MF) membrane with pore size of 0.01 m was used in Systen I of which filtration mode was set at constant pressure of $1kgf/cm^2$. Ultrafiltration (UF) membranes with molecular cutoff (MWCO) of 80,000 and 13,000 were used in System II-1 and II-2, respectively. Constant flow mode was applied at the range between 0.7 and $1.5m^3/m^2{\cdot}d$ (average of $1.1m^3/m^2${\cdot}d) for System II-1 and between 0.37 and $1.65m^3/m^2{\cdot}d$ (average of $1.18m^3/m^2{\cdot}d$) for System II-2. In System I, the flux changed from $1m^3/m^2{\cdot}d$ to $0.2m^3/m^2{\cdot}d$ during the operation time of 5 months. System II showed recovery of 94% under the allowable maximum pressure of $3kgf/cm^2$ during the same operation period. From these results, the efficient operation was observed in constant flow mode with respect to filtration time and recovery. Average filtrate turbidity showed 0.0071 NTU in System I and 0.0054 NTU in System II, which implied that high turbidity removal was obtained in both MF and UF systems with no significant difference between MF and UF. From the fact that membrane flux depends largely on membrane type and operation mode, a guideline of optimum design and operation should be suggested for application of membrane systems to full scale water treatment.

Design of Spinning and Subsequent Drawing Parameters to Improve the Mechanical Properties of PVA Fibers

  • Chae, Dong Wook;Kim, Seung Gyoo;Kim, Byoung Chul
    • Textile Coloration and Finishing
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    • v.28 no.3
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    • pp.125-133
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    • 2016
  • In this study, efforts were made to enhance the mechanical properties of the poly(vinyl alcohol) (PVA) fibers of medium molecular weight(number-average degree of polymerization=1735) varying the ratio in $DMSO/H_2O$ mixed solvent and spinning/drawing conditions. The gel fibers prepared from pure DMSO were opaquely frozen in the coagulating bath of $-20^{\circ}C$. However, transparent gel fibers were formed without freezing for the mixture to contain water less than 80wt%. As the amount of water in the mixture increased the residual solvent in the coagulated gel fibers decreased ranging from 85 to 42wt%. The complex viscosity increased with increasing PVA concentration in 80/20 $DMSO/H_2O$ exhibiting remarkable shear thinning at 18wt%. In the Cole-Cole plot, the 18wt% PVA solutions gave a deviated curve from 12 and 15wt% ones. Thus the optimum PVA concentration for the spinning processing of medium MW PVA solutions in 80/20 $DMSO/H_2O$ was determined to 18wt% with rheological concept. Low degree of drawing during hot drawing process in the dry state was available for high bath draft in the coagulation bath. The most improved mechanical properties were observed by applying the highest possible draw ratio attained by reducing bath draft over multi-step drawing process. In the given bath draft, linear relationship was observed between both tensile strength and modulus and draw ratio showing the inflection points at the draw ratio of 19.5 and 18.0 for tensile strength and modulus, respectively.

High Level Production of human Protein Tyrosine Kinase-6 in Insect Cells Using Drosophila Peptidoglycan Recognition Protein-LB as a fusion protein (곤충세포에서 새로운 퓨전 단백질인 초파리 유래 PGRP-LB를 이용한 인간 PTK6의 과발현 및 생산)

  • Kim, Seul-Ki;Kim, Han-Ie;Woo, Jae-Sung;Cho, Hyun-Soo;Jung, Yun-Jin;Lee, Seung-Taek;Ha, Nam-Chul
    • Journal of Life Science
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    • v.17 no.2 s.82
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    • pp.179-184
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    • 2007
  • PTK6, an intracellular protein tyrosine kinase, is significantly overexpressed in a majority of breast cancers and has a role in promoting the proliferation of the cancer cells, but not of normal cells. Here, we report high-level production of the catalytic unit of PTK6 fused with Drosophila peptidoglycan recognition protein (PGRT)-LB, in the baculovirus system. We first found that the PGRP-LB was potentially useful as a fusion partner to increase the yield of heterologous protein in the baculovirus system. The purified recombinant protein exhibited a 1.5-fold activity with much higher yield than the bacterially-expressed protein. The protein expressed in the baculovirus system will be useful for the crystallization to determine its crystal structure helping understand the molecular mechanism of PTK6 and design its inhibitors.

Crystallization and Preliminary X-Ray Crystallographic Analysis of UDP-N-Acetylglucosamine Enolpyruvyl Transferase from Haemophilus influenzae in Complex with UDP-N-Acetylglucosamine and Fosfomycin

  • Yoon, Hye-Jin;Ku, Min-Je;Ahn, Hyung Jun;Lee, Byung Il;Mikami, Bunzo;Suh, Se Won
    • Molecules and Cells
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    • v.19 no.3
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    • pp.398-401
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    • 2005
  • The bacterial enzyme UDP-N-acetylglucosamine enolpyruvyl transferase catalyzes the first committed step of peptidoglycan biosynthesis, i.e., transfer of enolpyruvate from phosphoenolpyruvate to UDP-N-acetyl-glucosamine. We have overexpressed the enzyme from Haemophilus influenzae in Escherichia coli and crystallized it in the apo-form, as well as in a complex with UDP-N-acetylglucosamine and fosfomycin using ammonium sulfate as the precipitant. X-ray diffraction data from a crystal of the apo-form were collected to $2.8{\AA}$ resolution at 293 K. The crystal quality was improved by co-crystallization with UDP-N-acetylglucosamine and fosfomycin. X-ray data to $2.2{\AA}$ have been collected at 100 K from a flash-frozen crystal of the complex. The complex crystals belong to the orthorhombic space group I222 (or $I2_12_12_1$) with unit-cell parameters of a = 63.7, b = 124.5, and $c=126.3{\AA}$. Assuming a monomer of the recombinant enzyme in the crystallographic asymmetric unit, the calculated Matthews parameter ($V_M$) is $2.71{\AA}^3Da^{-1}$ and solvent content is 54.6%.

Preparation and Characterization of Antioxidant Peptides from Fermented Goat Placenta

  • Hou, Yinchen;Zhou, Jiejing;Liu, Wangwang;Cheng, Yongxia;Wu, Li;Yang, Gongming
    • Food Science of Animal Resources
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    • v.34 no.6
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    • pp.769-776
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    • 2014
  • The goat placenta was fermented by Bacillus subtilis and the optimal fermentation parameters of strongest antioxidant capacity of peptides were obtained using response surface methodology (RSM). The effects of fermentation time, initial pH value and glucose content on the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging capacity of the goat peptides were well fitted to a quadric equation with high determination coefficients. According to the data analysis of design expert, the strongest DPPH radical scavenging capacity value was obtained with the following conditions: content of glucose was 2.23%, initial pH value was 7.00 and fermentation time was 32.15 h. The DPPH radical scavenging capacity commonly referring antioxidant activity showed a concentration dependency and increased with increasing peptide concentration. The effects of temperature and pH were assessed to determine the stability of antioxidant peptides prepared from goat placenta. Antioxidant peptides showed good stabilities when temperature was lower than $70^{\circ}C$. However, the antioxidant peptides lost antioxidant activities rapidly under alkaline and excessive acid condition. Ultrafiltration technique was performed to separate fermentation broth with different Mw (molecular weight). It was found that peptides in the range of < 3 KDa mainly accounted for the antioxidant activities.