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1
Matsuura, Kazunori, Yuriko Shiomi, Toshihumi Mizuta та Hiroshi Inaba. "Horseradish Peroxidase-Decorated Artificial Viral Capsid Constructed from β-Annulus Peptide via Interaction between His-Tag and Ni-NTA". Processes 8, № 11 (13 листопада 2020): 1455. http://dx.doi.org/10.3390/pr8111455.
Анотація:
Artificial construction of spherical protein assemblies has attracted considerable attention due to its potential use in nanocontainers, nanocarriers, and nanoreactors. In this work, we demonstrate a novel strategy to construct peptide nanocapsules (artificial viral capsids) decorated with enzymes via interactions between His-tag and Ni-NTA. A β-annulus peptide derived from the tomato bushy stunt virus was modified with Ni-NTA at the C-terminus, which is directed toward the exterior surface of the artificial viral capsid. The β-annulus peptide bearing Ni-NTA at the C-terminus self-assembled into capsids of about 50 nm in diameter. The Ni-NTA-displayed capsids were complexed with recombinant horseradish peroxidase (HRP) with a C-terminal His-tag which was expressed in Escherichia coli. The β-annulus peptide-HRP complex formed spherical assemblies whose sizes were 30–90 nm, with the ζ-potential revealing that the HRP was decorated on the outer surface of the capsid.
2
Fonda, Irena, Maja Kenig, Vladka Gaberc-Porekar, Primo Pristovaek, and Viktor Menart. "Attachment of Histidine Tags to Recombinant Tumor Necrosis Factor-Alpha Drastically Changes Its Properties." Scientific World JOURNAL 2 (2002): 1312–25. http://dx.doi.org/10.1100/tsw.2002.215.
Анотація:
When studying two different histidine tags attached to the N-termini of the trimeric cytokine tumor necrosis factor alpha (TNF), the biological activity — measured as cytotoxicity on the L-929 cell line — of both tagged proteins was drastically reduced. The longer His10 tag reduced cytotoxicity to approximately 16% and the shorter His7 tag to 6% of the activity of their nontagged counterparts. After removal of the tags, biological activities reverted to the expected normal values, which clearly shows the key role of the attached histidine tags in diminishing biological activity. Studies on the mechanism of these effects revealed no specific interactions and showed that even the natural flexible N-terminus of TNF presents a steric hindrance for receptor binding, while any extension of the N-terminus increases this hindrance and consequently reduces biological activity. Also, in other proteins, the ligand or substrate binding sites may be hindered by histidine tags, leading to wrong conclusions about biological activity or other properties of the proteins. Thus caution is advised when using His-tagged proteins directly in screening procedures or in research.
3
Legardinier, Sébastien, Jean-Claude Poirier, Danièle Klett, Yves Combarnous, and Claire Cahoreau. "Stability and biological activities of heterodimeric and single-chain equine LH/chorionic gonadotropin variants." Journal of Molecular Endocrinology 40, no. 4 (February 15, 2008): 185–98. http://dx.doi.org/10.1677/jme-07-0151.
Анотація:
Recombinant equine LH/chorionic gonadotropin (eLH/CG) was expressed in the baculovirus–Sf9 insect cell system either as a single-chain with the C-terminus of the β-subunit fused to the N-terminus of the α-subunit or as non-covalently linked heterodimers with or without a polyhistidine tag at various locations. All these non-covalently linked eLH/CG variants were secreted as stable heterodimers in the medium of infected Sf9 cells. To assess the influence of the presence and the position of polyhistidine tag on LH bioactivity, we expressed four non-covalently linked tagged heterodimeric eLH/CG variants that were secreted in threefold higher quantities than the single chain. Among them, only two exhibited full in vitro LH bioactivity, relative to untagged heterodimers, namely the one His-tagged at the N-terminus of α-subunit and the other at the C-terminus of the β-subunit both of which are amenable to nickel-affinity purification. Furthermore, single-chain eLH/CG was found to be N- and O-glycosylated but nevertheless less active in in vitro LH bioassays than natural eCG and heterodimeric recombinant eLH/CG. The thermal stability of natural and recombinant hormones was assessed by the initial rates of dissociation from 20 to 90 °C. Heterodimeric eLH/CG from Sf9 cells was found to be as stable as pituitary eLH and serum eCG (T1/2, 74–77 °C). Although Sf9 cells only elaborated short immature-type carbohydrate side chains on glycoproteins, recombinant eLH/CG produced in these cells exhibited stabilities similar to that of pituitary eLH. In conclusion, recombinant heterodimeric eLH/CG exhibits the same thermal stability as natural pituitary LH and its advantages over the single-chain eLH/CG include higher secretion, higher in vitro bioactivity, and reduced potential risk of immunogenicity.
4
Ilic-Tomic, Tatjana, Sandra Markovic, and Branka Vasiljevic. "Expression and purification of the Sgm protein from E. coli." Journal of the Serbian Chemical Society 70, no. 6 (2005): 817–22. http://dx.doi.org/10.2298/jsc0506817i.
Анотація:
The Sgm gene from Micromonospora zionensis, the producer of the aminoglycoside antibiotic G-52, encodes for Sgmmethylasewhich modifies the target site on 16S rRNAand thus protects the producer against its own toxic product. The sgm gene wasmodified by polymerase chain reaction (PCR) and cloned in the QIA express pQE-30 vector in order to make a construct that places the (His)6 tag at the N-terminus of the protein. The resulting expression construct was transformed in the E. coli strain NM522 and the functional activity of the Sgm-His fusion protein was confirmed in vivo. Purification of the (His)6-tagged Sgm protein by Ni-NTA affinity chromatography was performed under native conditions and the protein was detected on a sodium dodecyl sulfate polyacrylamide gel. Sgm methylase was purified to homogeneity > 95 %. Polyclonal antibodies raised to purified (His)6-tagged Sgm protein were used to identify this protein by Western blot analysis.
5
Ganguly, Advaita, Ravindra B. Malabadi, Dipankar Das, Mavanur R. Suresh, and Hoon H. Sunwoo. "Enhanced Prokaryotic Expression of Dengue Virus Envelope Protein." Journal of Pharmacy & Pharmaceutical Sciences 16, no. 4 (October 20, 2013): 609. http://dx.doi.org/10.18433/j3pc80.
Анотація:
Purpose. To highlight the expression and purification of the recombinant dengue virus type-1 antigen exploiting the codon optimized full length envelope for increased yield in E. coli. Methods. A 6x His tag was inserted at the C terminus to facilitate purification. The purified protein was recognized in Western blot by Monoclonal antibody specific for the tag. The in vitro refolded recombinant protein was used to immunize mice for the development of hybridomas and also analyzed for its biological functionality with heparan sulfate binding assay. Results. The polyclonal anti-sera from the immunized mice were found to recognize the envelope protein thereby establishing the immunogenicity of the protein. Conclusion. The purified envelope protein could potentially be used towards dengue diagnostics and vaccine development efforts. This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.
6
Perron-Savard, Philippe, Gregory De Crescenzo, and Hervé Le Moual. "Dimerization and DNA binding of the Salmonella enterica PhoP response regulator are phosphorylation independent." Microbiology 151, no. 12 (December 1, 2005): 3979–87. http://dx.doi.org/10.1099/mic.0.28236-0.
Анотація:
In Salmonella enterica, PhoP is the response regulator of the PhoP/PhoQ two-component regulatory system that controls the expression of various virulence factors in response to external Mg2+. Previous studies have shown that phosphorylation of a PhoP variant with a C-terminal His tag (PhoPHis) enhances dimerization and binding to target DNA. Here, the effect of phosphorylation on the oligomerization and DNA binding properties of both wild-type PhoP (PhoP) and PhoPHis are compared. Gel filtration chromatography showed that PhoP exists as a mixture of monomer and dimer regardless of its phosphorylation state. In contrast, unphosphorylated PhoPHis was mostly monomeric, whereas PhoPHis∼P existed as a mixture of monomer and dimer. By monitoring the tryptophan fluorescence of the proteins and the fluorescence of the probe 1-anilinonaphthalene-8-sulfonic acid bound to them, it was found that PhoP and PhoPHis exhibited different spectral properties. The interaction between PhoP or PhoPHis and the PhoP box of the mgtA promoter was monitored by surface plasmon resonance. Binding of PhoP to the PhoP box was barely influenced by phosphorylation. In contrast, phosphorylation of PhoPHis clearly increased the interaction of PhoPHis with target DNA. Altogether, these data show that a His tag at the C-terminus of PhoP affects its biochemical properties, most likely by affecting its conformation and/or its oligomerization state. More importantly, these results show that wild-type PhoP dimerization and interaction with target DNA are independent of phosphorylation, which is in contrast to the previously proposed model.
7
Schweida, David, Pierre Barraud, Christof Regl, Fionna E. Loughlin, Christian G. Huber, Chiara Cabrele, and Mario Schubert. "The NMR signature of gluconoylation: a frequent N-terminal modification of isotope-labeled proteins." Journal of Biomolecular NMR 73, no. 1-2 (February 8, 2019): 71–79. http://dx.doi.org/10.1007/s10858-019-00228-6.
Анотація:
Abstract N-terminal gluconoylation is a moderately widespread modification in recombinant proteins expressed in Escherichia coli, in particular in proteins bearing an N-terminal histidine-tag. This post-translational modification has been investigated mainly by mass spectrometry. Although its NMR signals must have been observed earlier in spectra of 13C/15N labeled proteins, their chemical shifts were not yet reported. Here we present the complete 1H and 13C chemical shift assignment of the N-terminal gluconoyl post-translational modification, based on a selection of His-tagged protein constructs (CCL2, hnRNP A1 and Lin28) starting with Met-Gly-...-(His)6. In addition, we show that the modification can hydrolyze over time, resulting in a free N-terminus and gluconate. This leads to the disappearance of the gluconoyl signals and the appearance of gluconate signals during the NMR measurements. The chemical shifts presented here can now be used as a reference for the identification of gluconoylation in recombinant proteins, in particular when isotopically labeled.
8
Kim, Jong Kyong, Scott B. Mulrooney, and Robert P. Hausinger. "The UreEF Fusion Protein Provides a Soluble and Functional Form of the UreF Urease Accessory Protein." Journal of Bacteriology 188, no. 24 (October 13, 2006): 8413–20. http://dx.doi.org/10.1128/jb.01265-06.
Анотація:
ABSTRACT Four accessory proteins (UreD, UreE, UreF, and UreG) are typically required to form the nickel-containing active site in the urease apoprotein (UreABC). Among the accessory proteins, UreD and UreF have been elusive targets for biochemical and structural characterization because they are not overproduced as soluble proteins. Using the best-studied urease system, in which the Klebsiella aerogenes genes are expressed in Escherichia coli, a translational fusion of ureE and ureF was generated. The UreEF fusion protein was overproduced as a soluble protein with a convenient tag involving the His-rich region of UreE. The fusion protein was able to form a UreD(EF)G-UreABC complex and to activate urease in vivo, and it interacted with UreD-UreABC in vitro to form a UreD(EF)-UreABC complex. While the UreF portion of UreEF is fully functional, the fusion significantly affected the role of the UreE portion by interrupting its dimerization and altering its metal binding properties compared to those of the wild-type UreE. Analysis of a series of UreEF deletion mutants revealed that the C terminus of UreF is required to form the UreD(EF)G-UreABC complex, while the N terminus of UreF is essential for activation of urease.
9
Mollaev, M. D., A. I. Zabolotskii, D. A. Mazalev, N. V. Gorokhovets, M. B. Sokol, M. R. Mollaeva, M. V. Fomicheva, A. B. Pshenichnikova, and E. D. Nikolskaya. "Obtaining and Purification of Recombinant Domain III of Human Alpha-Fetoprotein." Biotekhnologiya 37, no. 4 (2021): 32–42. http://dx.doi.org/10.21519/0234-2758-2021-37-4-32-42.
Анотація:
Previous studies have demonstrated the applicability of alpha-fetoprotein or its receptor-binding domain fused to the 6-histidine tag at the C-terminus (rAFP3D-His6) as vectors for targeted delivery of antitumor agents. This tag is undesirable for further preclinical trials. Therefore, we designed a recombinant protein rAFP3D without any affine tags, and accessed its functional activity. The protein was produced as inclusion bodies in Escherichia coli BL21 (DE3) cells. Optimal conditions for washing inclusion bodies and refolding the target protein were selected. We used a saturated solution of (NH4)2SO4 as the primary purification step to precipitate the main fraction of protein admixtures. The second purification step included hydrophobic chromatography using butyl-cellulose. The identity of the protein sequence was confirmed by tandem mass spectrometry. Circular dichroism demonstrated the authenticity of the secondary structure. Fluorescently labeled rAFP3D actively penetrated into MCF-7 tumor cells. These results indicate that rAFP3D can be used for targeted drug delivery. Key words: alpha-fetoprotein, receptor-binding domain, recombinant protein, MALDI-MS, HPLC, drug delivery system Funding - The project was supported with Russian Foundation for Basic Research (project no. 18-29-09022/20).
10
Sahlan, Muhamad, Budiman Bela, Anom Bowolaksono, Amarila Malik, and Masafumi Yohda. "THE EXPRESSION AND PURIFICATION OF OCTA-ARGININE APOPTIN AND ITS ABILITY TO KILL CANCER CELLS." International Journal of Pharmacy and Pharmaceutical Sciences 8, no. 10 (August 12, 2016): 102. http://dx.doi.org/10.22159/ijpps.2016v8i10.11455.
Анотація:
<p><strong>Objective: </strong>In this research, <em>chicken anemia virus</em> apoptin optimized genetically for expression in <em>Escherichia coli</em> and also modified using (His)<sub>6</sub> tag, (Arg)<sub>8</sub> tag, and HlyA tag intended for purification needs, penetration enhancement, and secretion from<em> </em>bacterial host<em> </em>to the growth media.</p><p><strong>Methods</strong><strong>:</strong><strong> </strong>The modified apoptin gene was optimized using an Integrated DNA Technology (IDT). The gene (606 bp) then ordered and synthesized by Eurofins. The apoptin gene was expressed using <em>E. coli</em> BL21 CodonPlus as host, in cultivation temperature of 37 °C, and 25 °C and purified using Ni-NTA agarose beads. The addition of (His)<sub> 6</sub> tag enabled the apoptin to be purified in only one step by using nickel column. The expression and purification data analyzed qualitatively as well as quantitatively using SDS-PAGE. MTT assay was used to identify the antitumor effect of octa arginine-apoptin to two kinds of cancer cells, cervix HeLa cancer cell and colon Widr cancer cell. The viability of cell was analyzed when the cell incubated in the variation concentration protein for 72 h.</p><p><strong>Results: </strong>The constructed apoptin gene were expressed in <em>E. coli </em>successfully. The MTT assay indicated that Octaarginin-Apoptin was able to induce apoptosis of HeLa and Widr cells lines in a dose-dependent manner. The recombinant apoptin without tagging with octa-arginine, have no ability to induce apoptosis of HeLa and Widr cells lines. <strong></strong></p><p class="Abstract"><strong>Conclusion: </strong><em>This octa arginine-apoptin may in the future allow the development of a therapeutic protein that is able to kill cancer cells specifically.</em></p>
11
Xue, You-Lin, Takuya Miyakawa, Yoriko Sawano, and Masaru Tanokura. "Crystallization and preliminary X-ray crystallographic analysis of dioscorin fromDioscorea japonica." Acta Crystallographica Section F Structural Biology and Crystallization Communications 68, no. 2 (January 26, 2012): 193–95. http://dx.doi.org/10.1107/s1744309111053723.
Анотація:
Dioscorin, the major tuber storage protein in yam, has been reported to possess carbonic anhydrase, trypsin inhibitor, angiotensin-converting enzyme (ACE) inhibitor, free-radical scavenger, dehydroascorbate reductase and monodehydroascorbate reductase activities. Recent research has also found that dioscorin can enhance immune modulationviathe toll-like receptor 4 (TLR-4) signal transduction pathway in RAW 264.7 cells, murine bone-marrow cells and human monocytesex vivo. Resolving the structure of dioscorin would help in better understanding its activities and would provide clues to understanding the mechanism of its multiple functions. The full-length protein (residues 1–246) with an additional His6tag at the N-terminus was expressed inEscherichia coliRosetta (DE3) cells. After His-tag cleavage and purification, the protein was crystallized by the sitting-drop vapour-diffusion method at 278 K. An X-ray diffraction data set was collected to a resolution of 2.11 Å using a synchrotron X-ray source. The crystal belonged to space groupC2221, with unit-cell parametersa= 83.5,b= 156.8,c = 83.6 Å, and was estimated to contain two protein molecules per asymmetric unit.
12
Kontkanen, Hanna, Ann Westerholm-Parvinen, Markku Saloheimo, Michael Bailey, Marjaana Rättö, Ismo Mattila, Marzia Mohsina, Nisse Kalkkinen, Tiina Nakari-Setälä, and Johanna Buchert. "Novel Coprinopsis cinerea Polyesterase That Hydrolyzes Cutin and Suberin." Applied and Environmental Microbiology 75, no. 7 (February 6, 2009): 2148–57. http://dx.doi.org/10.1128/aem.02103-08.
Анотація:
ABSTRACT Three cutinase gene-like genes from the basidiomycete Coprinopsis cinerea (Coprinus cinereus) found with a similarity search were cloned and expressed in Trichoderma reesei under the control of an inducible cbh1 promoter. The selected transformants of all three polyesterase constructs showed activity with p-nitrophenylbutyrate, used as a model substrate. The most promising transformant of the cutinase CC1G_09668.1 gene construct was cultivated in a laboratory fermentor, with a production yield of 1.4 g liter−l purified protein. The expressed cutinase (CcCUT1) was purified to homogeneity by immobilized metal affinity chromatography exploiting a C-terminal His tag. The N terminus of the enzyme was found to be blocked. The molecular mass of the purified enzyme was determined to be around 18.8 kDa by mass spectrometry. CcCUT1 had higher activity on shorter (C2 to C10) fatty acid esters of p-nitrophenol than on longer ones, and it also exhibited lipase activity. CcCUT1 had optimal activity between pH 7 and 8 but retained activity over a wide pH range. The enzyme retained 80% of its activity after 20 h of incubation at 50°C, but residual activity decreased sharply at 60°C. Microscopic analyses and determination of released hydrolysis products showed that the enzyme was able to depolymerize apple cutin and birch outer bark suberin.
13
Domashenko, Alevtina D., Jiang Zhu, Matthew Stein, and Stephen G. Emerson. "Transduction of TAT-NF-YA Fusion Protein into K562 Human Chronic Myelogenous Leukemia Cells Upregulates HOXB4 Activity." Blood 104, no. 11 (November 16, 2004): 3542. http://dx.doi.org/10.1182/blood.v104.11.3542.3542.
Анотація:
Abstract Protein transduction to redirect hematopoietic stem cell (HSC) fate for clinical therapy is an attractive alternative to genetic modification, in that the stem cell genome is not permanently modified. Indeed, published studies suggest that protein transduction with HOXB4, similar to retroviral overexpression of HoxB4, can increase HSC competitive repopulating ability. We have recently found that the trimeric transcription factor NF-Y, by modulation of its regulated subunit NF-YA, controls the expression of several HSC activating genes, including the Hox4 paralogs HoxB4, HoxC4, HoxD4 as well as p27, telomerase, Notch1, and LEF1. We have therefore now asked whether we can deliver NF-YA to hematopoietic cells by fusion with the protein transduction domain of the HIV-1 TAT protein, and modeled the molecular and biological activity of this protein by its effects on the HoxB4 promoter in vitro and in vivo. First, we designed and tested three fusion constructs expressing human NF-YA: with an N-terminal 6-histidine leader, a C-terminal 6xHis tag, and a GST tag at the N-terminus of NF-YA. Recombinant proteins bearing either the N- or C-terminal His-tag were poorly expressed under diverse growing and induction conditions, and were highly contaminated with bacterial host proteins. GST-fusion protein showed a much higher expression level and no host protein contamination, so all experiments were performed using GST-constructs. Next, electrophoretic mobility shift assays (EMSA) demonstrated that purified GST-TAT-NF-YA binds to HoxB4 promoter probes identically to endogenously translated NF-YA. NIH 3T3 fibroblasts, or K562 cells cultured in 0.1–2mM GST-TAT-NF-YA for 1.5h, but not without recombinant protein, showed bright cytoplasmic and nuclear fluorescence upon staining with anti-GST. To test the ability of GST-TAT-NF-YA to activate the HoxB4 promoter in live cells, GST-TAT-NF-YA’s effect was measured both on transfected HOXB4 promoter reporter constructs and on endogenous HoxB4 mRNA. First, when GST-TAT-NF-YA was incubated with K562 cells electroporated with HoxB4 promoter/luciferase, along with plasmids encoding the NF-Ya partners NF-YB, NF-YC, USF1, USF2 (and Renilla luciferase for transfection control), normalized luciferase activity calculated as a % relative to control HoxB4-luc activity increased in an NF-YA concentration-dependent fashion. More pronounced effects were seen when GST-TAT-NF-YA protein was added to stably transfected HOXB4-luc K562 cell lines, with up to 2.7 fold increased HoxB4-luc activity versus control cells. Importantly, addition of GST-TAT-NF-YA protein to unmanipulated K562 cells increased expression of endogenous HoxB4 mRNA 2-2.5X, as measured by real-time PCR. Taken together, these data demonstrate that NF-YA protein can be biochemically, efficiently and functionally delivered to hematopoietic cells via TAT-mediated technology, providing a promising alternative to gene transfer to stimulate multiple HSC-activating genes.
14
Yang, Jie, Junlei Zhang, Wei Chen, Zhen Hu, Junmin Zhu, Xin Fang, Wenchang Yuan, et al. "Eliciting cross-neutralizing antibodies in mice challenged with a dengue virus envelope domain III expressed inEscherichia coli." Canadian Journal of Microbiology 58, no. 4 (April 2012): 369–80. http://dx.doi.org/10.1139/w11-137.
Анотація:
Dengue viruses (DENVs) are mosquito-borne infectious pathogens that pose a serious global public health threat, and at present, no therapy or effective vaccines are available. Choosing suitable units as candidates is fundamental for the development of a dengue subunit vaccine. Domain III of the DENV-2 E protein (EDIII) was chosen in the present study and expressed in Escherichia coli by N-terminal fusion to a bacterial leader (pelB), and C-terminal fusion with a 6×His tag based on the functions of DENV structure proteins, especially the neutralizing epitopes on the envelope E protein. After two-step purification using Ni–NTA affinity and cation-exchange chromatography, the His-tagged EDIII was purified up to 98% homogenicity. This recombinant EDIII was able to trigger high levels of neutralizing antibodies in both BALB/c and C57BL/6 mice. Both the recombinant EDIII and its murine antibodies protected Vero cells from DENV-2 infection. Interestingly, the recombinant EDIII provides at least partial cross-protection against DENV-1 infection. In addition, the EDIII antibodies were able to protect suckling mice from virus challenge in vivo. These data suggest that a candidate molecule based on the small EDIII protein, which has neutralizing epitopes conserved among all 4 DENV serotypes, has important implications.
15
Hebrard, Eugenie, Martin Drucker, Denis Leclerc, Thomas Hohn, Marilyne Uzest, Remy Froissart, Jean-Marc Strub, et al. "Biochemical Characterization of the Helper Component of Cauliflower Mosaic Virus." Journal of Virology 75, no. 18 (September 15, 2001): 8538–46. http://dx.doi.org/10.1128/jvi.75.18.8538-8546.2001.
Анотація:
ABSTRACT The helper component of Cauliflower mosaic virus is encoded by viral gene II. This protein (P2) is dispensable for virus replication but required for aphid transmission. The purification of P2 has never been reported, and hence its biochemical properties are largely unknown. We produced the P2 protein via a recombinant baculovirus with a His tag fused at the N terminus. The fusion protein was purified by affinity chromatography in a soluble and biologically active form. Matrix-assisted laser desorption time-of-flight mass spectrometry demonstrated that P2 is not posttranslationally modified. UV circular dichroism revealed the secondary structure of P2 to be 23% α-helical. Most α-helices are suggested to be located in the C-terminal domain. Using size exclusion chromatography and aphid transmission testing, we established that the active form of P2 assembles as a huge soluble oligomer containing 200 to 300 subunits. We further showed that P2 can also polymerize as long paracrystalline filaments. We mapped P2 domains involved in P2 self-interaction, presumably through coiled-coil structures, one of which is proposed to form a parallel trimer. These regions have previously been reported to also interact with viral P3, another protein involved in aphid transmission. Possible interference between the two types of interaction is discussed with regard to the biological activity of P2.
16
Altendorf, K., W. Stalz, J. Greie, and G. Deckers-Hebestreit. "Structure and function of the F(o) complex of the ATP synthase from Escherichia coli." Journal of Experimental Biology 203, no. 1 (January 1, 2000): 19–28. http://dx.doi.org/10.1242/jeb.203.1.19.
Анотація:
The membrane-bound ATP synthase (F(1)F(o)) from mitochondria, chloroplasts and bacteria plays a crucial role in energy-transducing reactions. In the case of Escherichia coli, the reversible, proton-translocating ATPase complex consists of two different entities, F(1) and F(o). The water-soluble F(1) part carries the catalytic sites for ATP synthesis and hydrolysis. It is associated with the membrane-embedded F(o) complex, which functions as a proton channel and consists of subunits a, b and c present in a stoichiometry of 1:2:12.Subunit b was isolated by preparative gel electrophoresis, acetone-precipitated and renatured in a cholate-containing buffer. Reconstituted subunit b together with purified ac subcomplex is active in proton translocation and F(1) binding, thereby demonstrating that subunit b had recovered its native conformation. Circular dichroism spectroscopy of subunit b reconstituted into liposomes revealed a rather high degree of alpha -helical conformation of 80%. After addition of a His(6)-tag to the N terminus of subunit a, a stable ab(2) subcomplex was purified instead of a single subunit a, arguing in favour of a direct interaction between these subunits. After addition of subunit c and reconstitution into phospholipid vesicles, an F(o) complex was obtained exhibiting rates of proton translocation and F(1) binding comparable with those of wild-type F(o).The epitopes of monoclonal antibodies against subunit c are located in the hydrophilic loop region (cL31-Q42) as mapped by enzyme-linked immunosorbent assay using overlapping synthetic heptapeptides. Binding studies revealed that all monoclonal antibodies (mAbs) bind to everted membrane vesicles irrespective of the presence or absence of F(1). Although the hydrophilic region of subunit c, and especially the highly conserved residues cA40, cR41, cQ42 and cP43, are known to interact with subunits gamma and epsilon of the F(1) part, the mAb molecules have no effect on the function of F(o), either in proton translocation or in F(1) binding. However, the F(1) part and the mAb molecule(s) are bound simultaneously to the F(o) complex, suggesting that not all c subunits are involved in the interaction with F(1).
17
Robertson, D., H. F. Paterson, P. Adamson, A. Hall, and P. Monaghan. "Ultrastructural localization of ras-related proteins using epitope-tagged plasmids." Journal of Histochemistry & Cytochemistry 43, no. 5 (May 1995): 471–80. http://dx.doi.org/10.1177/43.5.7537292.
Анотація:
To determine the ultrastructural distribution of H-ras, the rho proteins rho-A, rho-B, rho-C, and the rac1 protein (members of the ras GTP-binding protein family), we used cDNA expression plasmids in which a short sequence coding for the epitope recognized by the anti c-myc monoclonal antibody 9E10 has been inserted at the N-terminus. Each of the expressed proteins has this epitope as a tag, allowing its localization by light and electron microscopy by the same antibody. After nuclear microinjection of these plasmids into MDCK or Rat 2 cells, expression of the protein (6-18 hr later) was confirmed by immunofluorescence labeling with 9E10 imaged by confocal microscopy. For ultrastructural localization of these tagged proteins, a method was devised to process microinjected cells in situ into low-temperature resin. The proteins were localized on the sections using 9E10 detected with colloidal gold conjugates. Ha-ras protein was localized almost exclusively on the cell membranes. Rho-A and rho-C were predominantly associated with the submembraneous actin network, and rho-B was found in association with multivesicular bodies. Rac1 protein induces the formation of large pinocytotic vesicles and was detected on the cytoplasmic face of these vacuoles. These experiments demonstrate the successful use of this approach for detection of de novo synthesized proteins from microinjected plasmids by both light and electron microscopy on a small (< 50 cells) sample size.
18
Story, Sherry V., Claudia Shah, Francis E. Jenney, and Michael W. W. Adams. "Characterization of a Novel Zinc-Containing, Lysine-Specific Aminopeptidase from the Hyperthermophilic Archaeon Pyrococcus furiosus." Journal of Bacteriology 187, no. 6 (March 15, 2005): 2077–83. http://dx.doi.org/10.1128/jb.187.6.2077-2083.2005.
Анотація:
ABSTRACT Cell extracts of the proteolytic, hyperthermophilic archaeon Pyrococcus furiosus contain high specific activity (11 U/mg) of lysine aminopeptidase (KAP), as measured by the hydrolysis of l-lysyl-p-nitroanilide (Lys-pNA). The enzyme was purified by multistep chromatography. KAP is a homotetramer (38.2 kDa per subunit) and, as purified, contains 2.0 ± 0.48 zinc atoms per subunit. Surprisingly, its activity was stimulated fourfold by the addition of Co2+ ions (0.2 mM). Optimal KAP activity with Lys-pNA as the substrate occurred at pH 8.0 and a temperature of 100°C. The enzyme had a narrow substrate specificity with di-, tri-, and tetrapeptides, and it hydrolyzed only basic N-terminal residues at high rates. Mass spectroscopy analysis of the purified enzyme was used to identify, in the P. furiosus genome database, a gene (PF1861) that encodes a product corresponding to 346 amino acids. The recombinant protein containing a polyhistidine tag at the N terminus was produced in Escherichia coli and purified using affinity chromatography. Its properties, including molecular mass, metal ion dependence, and pH and temperature optima for catalysis, were indistinguishable from those of the native form, although the thermostability of the recombinant form was dramatically lower than that of the native enzyme (half-life of approximately 6 h at 100°C). Based on its amino acid sequence, KAP is part of the M18 family of peptidases and represents the first prokaryotic member of this family. KAP is also the first lysine-specific aminopeptidase to be purified from an archaeon.
19
Markovic, Sandra, Sandra Vojnovic, Milija Jovanovic, and Branka Vasiljevic. "Different expression levels of two KgmB-His fusion proteins." Journal of the Serbian Chemical Society 70, no. 12 (2005): 1401–7. http://dx.doi.org/10.2298/jsc0512401m.
Анотація:
The KgmB methylase from Streptomyces tenebrarius was expressed and purified using the QIAexpress System. Two expression vectors were made: pQEK-N, which places a (His)6 tag at the N-terminus, and pQEK-C, which places a (His)6 tag at the C-terminus of the recombinant KgmB protein. Kanamycin resistance of the E. coli cells containing either the pQEK-N or the pQEK-C recombinant plasmids confirmed the functionality of both KgmB-His fusion proteins in vivo. Interestingly, different levels of expression were observed between these two recombinant proteins. Namely, KgmB methylase with the (His)6 tag at the N-terminus showed a higher level of expression. Purification of the (His)6-tagged proteins using Ni-NTA affinity chromatography was performed under native conditions and the KgmB methylase with (His)6 tag at the N-terminus was purified to homogeneity >95 %. The recombinant KgmB protein was detected on a Western blot using anti-Sgm antibodies.
20
Gao, Weiqiang, Patricia J. Anderson, Elaine M. Majerus, Elodee A. Tuley, and J. Evan Sadler. "A C-Terminal Exosite of von Willebrand Factor Domain A2 Interacts with ADAMTS13 Spacer Domain To Accelerate Substrate Cleavage." Blood 108, no. 11 (November 16, 2006): 1785. http://dx.doi.org/10.1182/blood.v108.11.1785.1785.
Анотація:
Abstract ADAMTS13 is a plasma metalloproteinase that limits platelet-rich thrombi by cleaving von Willebrand factor (VWF) at the Tyr1605-Met1606 bond in the A2 domain. A minimal substrate that consists of GST linked to VWF residues Asp1596-Arg1668 with a C-terminal 6×His tag (GST-VWF73) is cleaved rapidly by plasma ADAMTS13. Further removal of Glu1660-Arg1668, which comprises a predicted C-terminal α-helix (GST-VWF64) markedly reduced the rate of cleavage, suggesting this helix comprises an exosite for substrate recognition. By amino acid sequencing, ADAMTS13 was shown to cleave the Tyr1605-Met1606 bond of GST-VWF64. Truncation of ADAMTS13 after certain structural domains had different effects on substrate cleavage. After removal of the GST moiety by proteolysis, the kinetic constants for cleavage of VWF73 and VWF64 were determined for several recombinant ADAMTS13 variants using a quantitative MALDI-MS assay (Table). Comparison of the specificity constants (kcat/Km) shows that ADAMTS13 truncated after the spacer domain (construct MDTCS) cleaved VWF73 ~20-fold faster than did a similar enzyme without the spacer domain (construct MDTC). In contrast, both MDTCS and MDTC cleaved VWF64 slowly at a rate similar to the cleavage of VWF73 by MDTC. Most of the variation in cleavage rates was explained by differences in Km, suggesting that the spacer domain recognizes an exosite at the C-terminus of VWF73 that is missing from VWF64. Cleavage of VWF73 yields a C-terminal product (cVWF63). Purified cVWF63 (7.5 μM) inhibited MDTCS activity toward VWF73 or VWF64 (1.5 μM) by »90%, but did not inhibit MDTC, suggesting that the exosite of VWF73 or cVWF63 interacts directly with the spacer domain. Moreover, cVWF63 inhibited the cleavage of multimeric VWF by full-length ADAMTS13 and MDTCS up to 80%, but did not inhibit MDTC. The selective effects of deleting the ADAMTS13 spacer domain and Glu1660-Arg1668 of the VWF domain A2 suggest that the C-terminal exosite of the VWF A2 domain accelerates substrate cleavage by binding specifically to the ADAMTS13 spacer domain. Table Kinetics studies of VWF73 and VWF64 cleavage by ADAMTS13 and its truncations VWF73 VWF64 Enzyme K m (μM) k cat (s−1) k cat /K m (×105 M−1 s−1) K m (μM) k cat (s−1) k cat /K m (×105 M−1 s−1) FL-ADAMTS13 1.7±0.4 1.3±0.1 7.5±2.0 37.7±12.8 1.9±0.4 0.5±0.3 MDTCS 0.8±0.2 1.7±0.1 20.5±6.6 5.5±1.4 0.6±0.1 1.0±0.3 MDTC 16.0±4.5 1.8±0.3 1.1±0.5 17.9±6.3 1.6±0.3 0.9±0.5
21
Noguchi, Taro Q. P., Noriko Kanzaki, Hironori Ueno, Keiko Hirose, and Taro Q. P. Uyeda. "A Novel System for Expressing Toxic Actin Mutants in Dictyostelium and Purification and Characterization of a Dominant Lethal Yeast Actin Mutant." Journal of Biological Chemistry 282, no. 38 (July 26, 2007): 27721–27. http://dx.doi.org/10.1074/jbc.m703165200.
Анотація:
We have developed a novel system for expressing recombinant actin in Dictyostelium. In this system, the C terminus of actin is fused to thymosin β via a glycine-based linker. The fusion protein is purified using a His tag attached to the thymosin β moiety and then cleaved by chymotrypsin immediately after the native final residue of actin to yield intact actin. Wild-type actin prepared in this way was functionally normal in terms of its polymerization kinetics and muscle myosin-mediated motility. We expected that this system would be particularly useful for expressing toxic actin mutants, because the actin moiety of the fusion protein is unlikely to interact with the actin cytoskeleton of the host cells. We therefore chose to express the E206A/R207A/E208A mutant, which appears to be dominant lethal in yeast, as a model case of a toxic actin mutant that is difficult to express. We found that the E206A/R207A/E208A mutant could be expressed and purified with a yield comparable to the wild-type molecule (3–4 mg/20 g cells), even though green fluorescent protein-fused actin carrying the E206A/R207A/E208A mutation was expressed at a much lower level than wild-type actin. Purified E206A/R207A/E208A actin did not polymerize, even in the presence of muscle actin; however, it accelerated polymerization of muscle actin and inhibited the nucleating and severing activities of gelsolin. Given that the location of the substituted residues is near the pointed end face of the mutant, we suggest that E206A/R207A/E208A actin behaves like a weak pointed end-capping protein that perturbs the actin cytoskeleton of the host cells.
22
Wątły, J., A. Hecel, R. Wieczorek, J. Świątek-Kozłowska, H. Kozłowski, and M. Rowińska-Żyrek. "Uncapping the N-terminus of a ubiquitous His-tag peptide enhances its Cu2+ binding affinity." Dalton Transactions 48, no. 36 (2019): 13567–79. http://dx.doi.org/10.1039/c9dt01635j.
23
Zhang, Huang-Ge, Jinfu Xie, Igor Dmitriev, Elena Kashentseva, David T. Curiel, Hui-Chen Hsu, and John D. Mountz. "Addition of Six-His-Tagged Peptide to the C Terminus of Adeno-Associated Virus VP3 Does Not Affect Viral Tropism or Production." Journal of Virology 76, no. 23 (December 1, 2002): 12023–31. http://dx.doi.org/10.1128/jvi.76.23.12023-12031.2002.
Анотація:
ABSTRACT Production of large quantities of recombinant adeno-associated virus (AAV) is difficult and not cost-effective. To overcome this problem, we have explored the feasibility of creating a recombinant AAV encoding a 6×His tag on the VP3 capsid protein. We generated a plasmid vector containing a six-His (6×His)-tagged AAV VP3. A second plasmid vector was generated that contained the full-length AAV capsid capable of producing VP1 and VP2, but not VP3 due to a mutation at position 2809 that encodes the start codon for VP3. These plasmids, necessary for production of AAV, were transfected into 293 cells to generate a 6×His-tagged VP3mutant recombinant AAV. The 6×His-tagged VP3 did not affect the formation of AAV virus, and the physical properties of the 6×His-modified AAV were equivalent to those of wild-type particles. The 6×His-tagged AAV did not affect the production titer of recombinant AAV and could be used to purify the recombinant AAV using an Ni-nitrilotriacetic acid column. Addition of the 6×His tag did not alter the viral tropism compared to wild-type AAV. These observations demonstrate the feasibility of producing high-titer AAV containing a 6×His-tagged AAV VP3 capsid protein and to utilize the 6×His-tagged VP3 capsid to achieve high-affinity purification of this recombinant AAV.
24
Akeboshi, Hiromi, Yasunori Chiba, Yoshiko Kasahara, Minako Takashiba, Yuki Takaoka, Mai Ohsawa, Youichi Tajima та ін. "Production of Recombinant β-Hexosaminidase A, a Potential Enzyme for Replacement Therapy for Tay-Sachs and Sandhoff Diseases, in the Methylotrophic Yeast Ogataea minuta". Applied and Environmental Microbiology 73, № 15 (8 червня 2007): 4805–12. http://dx.doi.org/10.1128/aem.00463-07.
Анотація:
ABSTRACT Human β-hexosaminidase A (HexA) is a heterodimeric glycoprotein composed of α- and β-subunits that degrades GM2 gangliosides in lysosomes. GM2 gangliosidosis is a lysosomal storage disease in which an inherited deficiency of HexA causes the accumulation of GM2 gangliosides. In order to prepare a large amount of HexA for a treatment based on enzyme replacement therapy (ERT), recombinant HexA was produced in the methylotrophic yeast Ogataea minuta instead of in mammalian cells, which are commonly used to produce recombinant enzymes for ERT. The problem of antigenicity due to differences in N-glycan structures between mammalian and yeast glycoproteins was potentially resolved by using α-1,6-mannosyltransferase-deficient (och1Δ) yeast as the host. Genes encoding the α- and β-subunits of HexA were integrated into the yeast cell, and the heterodimer was expressed together with its isozymes HexS (αα) and HexB (ββ). A total of 57 mg of β-hexosaminidase isozymes, of which 13 mg was HexA (αβ), was produced per liter of medium. HexA was purified with immobilized metal affinity column for the His tag attached to the β-subunit. The purified HexA was treated with α-mannosidase to expose mannose-6-phosphate (M6P) residues on the N-glycans. The specific activities of HexA and M6P-exposed HexA (M6PHexA) for the artificial substrate 4MU-GlcNAc were 1.2 ± 0.1 and 1.7 ± 0.3 mmol/h/mg, respectively. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern suggested a C-terminal truncation in the β-subunit of the recombinant protein. M6PHexA was incorporated dose dependently into GM2 gangliosidosis patient-derived fibroblasts via M6P receptors on the cell surface, and degradation of accumulated GM2 ganglioside was observed.
25
Karasawa, Tadahiro, Xingmin Wang, Tsuneo Maegawa, Yoshio Michiwa, Hiroyuki Kita, Koichi Miwa, and Shinichi Nakamura. "Clostridium sordellii Phospholipase C: Gene Cloning and Comparison of Enzymatic and Biological Activities with Those of Clostridium perfringens and Clostridium bifermentans Phospholipase C." Infection and Immunity 71, no. 2 (February 2003): 641–46. http://dx.doi.org/10.1128/iai.71.2.641-646.2003.
Анотація:
ABSTRACT The gene encoding Clostridium sordellii phospholipase C (Csp) was cloned and expressed as a histidine-tagged (His-tag) protein, and the protein was purified to compare its enzymatic and biological activities with those of Clostridium perfringens phospholipase C (Cpa) and Clostridium bifermentans phospholipase C (Cbp). Csp was found to consist of 371 amino acid residues in the mature form and to be more homologous to Cbp than to Cpa. The egg yolk phospholipid hydrolysis activity of the His-tag Csp was about one-third of that of His-tag Cpa, but the hemolytic activity was less than 1% of that of His-tag Cpa. His-tag Csp was nontoxic to mice. Immunization of mice with His-tag Cbp or His-tag Csp did not provide effective protection against the lethal activity of His-tag Cpa. These results indicate that Csp possesses similar molecular properties to Cbp and suggest that comparative analysis of toxic and nontoxic clostridial phospholipases is helpful for characterization of the toxic properties of clostridial phospholipases.
26
Dabrowski, S., and J. Kur. "Recombinant His-tagged DNA polymerase. II. Cloning and purification of Thermus aquaticus recombinant DNA polymerase (Stoffel fragment)." Acta Biochimica Polonica 45, no. 3 (September 30, 1998): 661–67. http://dx.doi.org/10.18388/abp.1998_4204.
Анотація:
The Stoffel DNA fragment, shortened by 12 bp from 5' end, coding for Stoffel DNA polymerase (missing 4 amino acids at N-terminus of Stoffel amino-acids sequence) from the thermophilic Thermus aquaticus (strain YT-1) was amplified, cloned and expressed in Escherichia coli. The recombinant Stoffel fragment contained a polyhistidine tag at the N-terminus (21 additional amino acids) that allowed its single-step isolation by Ni2+ affinity chromatography. The enzyme was characterized and displayed high DNA polymerase activity and thermostability evidently higher than the native Taq DNA polymerase.
27
CHEN, GEN-HUNG, LI-JUNG YIN, I.-HUA CHIANG, and SHANN-TZONG JIANG. "Cloning and Expression of Antibacterial Goat Lactoferricin from Escherichia coli AD494(DE3)pLysS Expression System." Journal of Food Protection 71, no. 12 (December 1, 2008): 2523–25. http://dx.doi.org/10.4315/0362-028x-71.12.2523.
Анотація:
Goat lactoferricin (GLfcin), an antibacterial peptide, is released from the N terminus of goat lactoferrin by pepsin digestion. Two GLfcin-related cDNAs, GLfcin L and GLfcin S, encoding Ala20-Ser60 and Ser36-Ser60 of goat lactoferrin, respectively, were cloned into the pET-23a(+) expression vector upstream from (His)6-Tag gene and transformed into Escherichia coli AD494(DE3)pLysS expression host. After being induced by isopropyl-β-d -thiogalactopyranoside (IPTG), two (His)6-Tag fused recombinant lactoferricins, GLfcin L-His·Tag and GLfcin S-His·Tag, were expressed in soluble form within the E. coli cytoplasm. The GLfcin L-His·Tag and GLfcin S-His·Tag were purified using HisTrap affinity chromatography. According to an antibacterial activity assay using the agar diffusion method, GLfcin L-His·Tag had antibacterial activity against E. coli BCRC 11549, Staphylococcus aureus BCRC 25923, and Propionibacterium acnes BCRC 10723, while GLfcin S-His·Tag was able to inhibit the growth of E. coli BCRC 11549 and P. acnes BCRC 10723. These two recombinant lactoferricins behaved as thermostable peptides, which could retain their activity for up to 30 min of exposure at 100°C.
28
Krachmarova, Elena, Milena Tileva, Elena Lilkova, Peicho Petkov, Klaus Maskos, Nevena Ilieva, Ivan Ivanov, Leandar Litov, and Genoveva Nacheva. "His-FLAG Tag as a Fusion Partner of Glycosylated Human Interferon-Gamma and Its Mutant: Gain or Loss?" BioMed Research International 2017 (2017): 1–12. http://dx.doi.org/10.1155/2017/3018608.
Анотація:
In order to obtain glycosylated human interferon-gamma (hIFNγ) and its highly prone to aggregation mutant K88Q, a secretory expression in insect cells was employed. To facilitate recombinant proteins purification, detection, and stability the baculovirus expression vectors were constructed to bear N-terminal His6-FLAG tag. Although the obtained proteins were glycosylated, we found that their biological activity was 100 times lower than expected. Our attempts to recover the biological properties of both proteins by tag removal failed due to enterokinase resistance of the tag. Surprisingly, the tag was easily cleaved when the proteins were expressed inE. colicells and the tag-free proteins showed fully restored activity. To shed light on this phenomenon we performed molecular dynamics simulations. The latter showed that the tags interact with the receptor binding domains and the flexible C-termini of the fusion proteins thus suppressing their complex formation with the hIFNγreceptor. We hypothesize that in the case of glycosylated proteins the tag/C-terminal interaction positions the FLAG peptide in close proximity to the glycans thus sterically impeding the enterokinase access to its recognition site.
29
Birchfield, Aaron S., and Cecilia A. McIntosh. "The Effect of Recombinant Tags on Citrus paradisi Flavonol-Specific 3-O Glucosyltransferase Activity." Plants 9, no. 3 (March 24, 2020): 402. http://dx.doi.org/10.3390/plants9030402.
Анотація:
Recombinant tags are used extensively in protein expression systems to allow purification through IMAC (Immobilized Metal Affinity Chromatography), identification through Western blot, and to facilitate crystal formation for structural analysis. While widely used, their role in enzyme characterization has raised concerns with respect to potential impact on activity. In this study, a flavonol-specific 3-O glucosyltransferase (Cp3GT) from grapefruit (Citrus paradisi) was expressed in Pichia pastoris, and was assayed in its untagged form and with a C-terminal c-myc/6x His tag under various conditions to determine the effect of tags. Prior characterization of pH optima for Cp3GT obtained through expression in Escherichia coli, containing an N-terminal thioredoxin/6x His tag, indicated an optimal pH of 7–7.5, which is indicative of a normal physiological pH and agrees with other glucosyltransferase (GT) pH optima. However, characterization of Cp3GT expressed using P. pastoris with a C-terminal c-myc-6x His tag showed a higher optimal pH of 8.5–9. This suggests a possible tag effect or an effect related to physiological differences between the cell expression systems. Results testing recombinant Cp3GT expressed in Pichia with and without C-terminal tags showed a possible tag effect with regard to substrate preference and interactions with metals, but no apparent effect on enzymatic kinetics or pH optima.
30
Jeevan, A., C. T. McFarland, T. Yoshimura, T. Skwor, H. Cho, T. Lasco, and D. N. McMurray. "Production and Characterization of Guinea Pig Recombinant Gamma Interferon and Its Effect on Macrophage Activation." Infection and Immunity 74, no. 1 (January 2006): 213–24. http://dx.doi.org/10.1128/iai.74.1.213-224.2006.
Анотація:
ABSTRACT Gamma interferon (IFN-γ) plays a critical role in the protective immune responses against mycobacteria. We previously cloned a cDNA coding for guinea pig IFN-γ (gpIFN-γ) and reported that BCG vaccination induced a significant increase in the IFN-γ mRNA expression in guinea pig cells in response to living mycobacteria and that the virulent H37Rv strain of Mycobacterium tuberculosis stimulated less IFN-γ mRNA than did the attenuated H37Ra strain. In this study, we successfully expressed and characterized recombinant gpIFN-γ with a histidine tag at the N terminus (His-tagged rgpIFN-γ) in Escherichia coli. rgpIFN-γ was identified as an 18-kDa band in the insoluble fraction; therefore, the protein was purified under denaturing conditions and renatured. N-terminal amino acid sequencing of the recombinant protein yielded the sequence corresponding to the N terminus of His-tagged gpIFN-γ. The recombinant protein upregulated major histocompatibility complex class II expression in peritoneal macrophages. The antiviral activity of rgpIFN-γ was demonstrated with a guinea pig fibroblast cell line (104C1) infected with encephalomyocarditis virus. Interestingly, peritoneal macrophages treated with rgpIFN-γ did not produce any nitric oxide but did produce hydrogen peroxide and suppressed the intracellular growth of mycobacteria. Furthermore, rgpIFN-γ induced morphological alterations in cultured macrophages. Thus, biologically active rgpIFN-γ has been successfully produced and characterized in our laboratory. The study of rgpIFN-γ will further increase our understanding of the cellular and molecular responses induced by BCG vaccination in the guinea pig model of pulmonary tuberculosis.
31
Besford, R. T., B. Thomas, N. S. Huskisson, and G. W. Butcher. "Characterization of Conformers of D 1 of Photosystem II Using Site-Directed Antibodies." Zeitschrift für Naturforschung C 45, no. 6 (June 1, 1990): 621–26. http://dx.doi.org/10.1515/znc-1990-0610.
Анотація:
Abstract Antibodies have been raised to synthetic peptides, corresponding to a region in the loop spanning helices 4 and 5 of D 1 protein (Ala 250-Phe 265) and to a region anticipated to be near the C terminus of mature D 1 (His 332-Ala 345). Polyclonal antibodies to the sequence His 332-Ala 345 reacted with a 32 kDa polypeptide in thylakoid preparations identified as D 1 from its resistance (pea) or susceptibility (wheat) to lysine-C degradation. A monoclonal antibody to His 332-Ala 345 reacted preferentially with a faster migrating polypeptide in SDS electrophoresis, a putative conformer of D 1. Polyclonal antibodies to the sequence Ala 250- Phe 265 also reacted with the faster running polypeptide but not with the population of molecules running at 32 kDa. The putative conformer of D 1 from wheat appears to be more resistant than the main D1 population to lysine-C degradation. Peptide analyses by Takahashi et al. [(1988) FEBS Lett, 240, 6 - 8 ] suggest Asn 335-Ala 344 lies at the processed C terminus. The present report provides immunological confirmation that this sequence is retained in mature D 1.
32
Gunawan, Setiyo, Hakun Wirawasista Aparamarta, Fadlilatul Taufany, Arief Prayogo, Shelma Hajar Anugrah Putri, and Christian Julius Wijaya. "Separation and purification of triglyceride from nyamplung (Calophyllum inophyllum) seed oil as biodiesel feedstock by using continuous countercurrent extraction." Malaysian Journal of Fundamental and Applied Sciences 16, no. 1 (February 2, 2020): 18–22. http://dx.doi.org/10.11113/mjfas.v16n1.1439.
Анотація:
Calophyllum inophyllum or commonly called nyamplung in Indonesia has the potential to be used as a biodiesel feedstock due to its high oil content. The purpose of this study was to determine the effect of feed flowrate and solvent to oil mass ratio on triglycerides (TAG) and free fatty acid (FFA) contents in non-polar lipid fraction (NPLF) of C. inophyllum seed oil by using continuous counter-current extraction. The contents of TAG and FFA in NPLF of C. inophyllum seed oil each sample points in continuous countercurrent extraction equipment. It was expected that the TAG became purer and suitable for biodiesel purpose. Various factors applied in this study were the effect of solvent to oil mass ratio and the effect of n-hexane + oil to methanol feed flowrate in liter per hour. Crude C. inophyllum seed oil contained 63.91% TAG, 15.76% FFA, 12.25% monoglycerides (MAG), dan 4.66% diglycerides (DAG). Separation and purification of TAG were carried out by using a packed column with the principle of countercurrent flow using n-hexane and methanol technical grade as solvents. The product of extraction analyzed TAG content qualitatively by using TLC and quantitatively using HT-GC, while NPLF from each sample points were analyzed using HT-GC. The optimum results were obtained by using a solvent to oil mass ratio of 6 (w:w) and (n-hexane + oil) to methanol feed flowrate of 6:15 (L/h:L/h) with 92.85% content of TAG, 2.19% content of FFA and 74.79% yield of NPLF.
33
Skomal, Gregory B., Grayson Wood, and Nick Caloyianis. "Archival tagging of a basking shark, Cetorhinus maximus, in the western North Atlantic." Journal of the Marine Biological Association of the United Kingdom 84, no. 4 (August 2004): 795–99. http://dx.doi.org/10.1017/s0025315404009968h.
Анотація:
A 6·1-m long female basking shark (Cetorhinus maximus) was tagged 73 km east of Nantucket Island, Massachusetts on 27 September 2001 with a pop-up archival transmitting tag. The tag detached prematurely on 6 December 2001 in an area approximately 800 km south-west of the tag site off the coast of North Carolina. The basking shark was vertically active for the 71-d tracking period, moving through depths and temperatures ranging from the surface to 320 m and 5·8 to 21·0°C, respectively. The shark displayed temporal variation in its residence depth and exhibited a marked temperature preference, with 72% of the temperature observations between 15·0 and 17·5°C. This track provides evidence that the basking shark associates with the continental shelf and shelf edge off the south-eastern United States during autumn. Moreover, it corroborates previous studies indicating that the basking shark remains active and does not hibernate during autumn.
34
Zhang, Weirui, David Motto, and David Ginsburg. "ADAMTS13 Binds VWF Via its C-Terminal CUB2 Domain." Blood 104, no. 11 (November 16, 2004): 126. http://dx.doi.org/10.1182/blood.v104.11.126.126.
Анотація:
Abstract Thrombotic thrombocytopenic purpura (TTP) is a life threatening illness due to a deficiency of the VWF-cleaving protease, ADAMTS13. The ADAMTS13 protein is composed of a propeptide, followed by a typical zinc metalloprotease domain. The C-terminal 2/3 of the molecule contains disintegrin-like, cystine-rich, and spacer domains, as well as a total of eight TSP1 motifs and two CUB domains. The function of this C-terminal portion of the molecule and its composite motifs is unknown, though TSP1 and CUB domains of other proteins have been shown to mediate protein-protein interactions. To further explore the interaction between ADAMTS13 and VWF, we cloned full length human cDNAs for both ADAMTS13 and VWF into the mammalian expression vector pcDNA3.1. These constructs were transiently transfected into 293T cells and COS cells respectively, and conditioned media collected for analysis. Using an anti-myc antibody, myc-tagged VWF co-immunoprecipitated (co-IP) with ADAMTS13, as demonstrated by western blot analysis using antisera raised against a C-terminal peptide derived from the predicted ADAMTS13 sequence. This direct interaction required partial denaturation of VWF in 1M urea, with no co-IP observed in the absence of urea. To map the segment within ADAMTS13 responsible for VWF binding, we cloned a series of overlapping ADAMTS13 fragments into the bacterial expression vector, Pet44b. Fusion proteins were purified by binding of the included His-tag to Ni-NTA beads and incubated with recombinant myc-VWF in the presence of 1M urea. Association with VWF was analyzed by co-IP with anti-myc followed by western blot analysis using an antibody to the C-terminal HSV-tag present in each fusion protein. The CUB2 (Glu1298- Thr1427) fusion protein co-IP’d with full-length VWF and also demonstrated concentration-dependent competition with full-length ADAMTS13 for VWF binding. In summary, we have demonstrated a direct protein-protein interaction between VWF and ADAMTS13. Binding requires partial denaturation of VWF and appears to be mediated primarily through contacts with the ADAMTS13 CUB2 domain. This interaction may account for the previously observed co-purification of VWF and ADAMTS13 from human plasma. Furthermore, the requirement for 1M urea suggests that this interaction may only occur physiologically under conditions of high shear. Though others have shown that the C-terminal domains of ADAMTS13, including CUB2, are not required for VWF cleavage in vitro, our data, together with several C-terminal mutations previously reported in TTP patients, suggest that interactions between VWF and the ADAMTS13 CUB2 domain may be important in vivo.
35
France, Peter. "Scott Moncrieff's First Translation." Translation and Literature 21, no. 3 (November 2012): 364–82. http://dx.doi.org/10.3366/tal.2012.0088.
Анотація:
C. K. Scott Moncrieff, famous as the translator of Proust, began his translating career in 1918 with La Chanson de Roland. Knowing nothing of Old French, he encountered this classic text while recovering from a war wound; the work of translation was a ‘solace’ in time of war, but also a homage to his friend Wilfred Owen and others who had ‘met their Rencesvals’ as the war drew to a close. Scott Moncrieff was no jingoist, but against the cynicism of Siegfried Sassoon's war poetry, he used the Old French epic to celebrate the positive values embodied in the idea of vassalage. Like his Proust, his Song of Roland sought to bring another world to life in English-speaking culture, in all its specific difference. Here this led him to adopt an archaizing and purportedly oral style, notably in the imitation of the assonanced laisses of the original.
36
NALASKOWSKI, Marcus M., Christina DESCHERMEIER, Werner FANICK, and Georg W. MAYR. "The human homologue of yeast ArgRIII protein is an inositol phosphate multikinase with predominantly nuclear localization." Biochemical Journal 366, no. 2 (September 1, 2002): 549–56. http://dx.doi.org/10.1042/bj20020327.
Анотація:
The function of the transcription regulator ArgRIII in the expression of several genes involved in the metabolism of arginine in yeast has been well studied. It was previously reported that it is also an inositol phosphate multikinase and an important factor of the mRNA export pathway [reviewed by Shears (2000) Bioessays 22, 786–789]. In the present study we report the cloning of a full-length 1248-bp cDNA encoding a human inositol phosphate multikinase (IPMK). This protein has a calculated molecular mass of 47.219kDa. Functionally important motifs [inositol phosphate-binding site, ATP-binding site, catalytically important SSLL (Ser-Ser-Leu-Leu) domain] are conserved between the human IPMK and yeast ArgRIII. Bacterially expressed protein demonstrated an inositol phosphate multikinase activity similar to that of yeast ArgRIII. Ins(1,4,5)P3 is phosphorylated at positions 3 and 6 up to Ins(1,3,4,5,6)P5. The human IPMK fused with a fluorescent protein tag is localized predominantly in the nucleus when transiently expressed in mammalian cells. A basic cluster in the protein's C-terminus is positively involved in nuclear targeting. These findings are consistent with the concept of a nuclear inositol phosphate signalling and phosphorylation pathway in mammalian cells.
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Hirsch, Andrea, Eirini Meimaridou, Monica Fernandez-Cancio, Amit V. Pandey, María Clemente, Laura Audi, Adrian J. L. Clark, and Christa E. Flück. "Loss of the C Terminus of Melanocortin Receptor 2 (MC2R) Results in Impaired Cell Surface Expression and ACTH Insensitivity." Endocrine Reviews 31, no. 6 (December 1, 2010): 943. http://dx.doi.org/10.1210/edrv.31.6.9987.
Анотація:
Objective Mutations in melanocortin receptor 2 (MC2R) and its related melanocortin receptor accessory protein (MRAP) cause familial glucocorticoid deficiency. We identified a novel MC2R mutation, K289fs. This unique mutation in the C terminus of MC2R is located in the intracellular part of the protein for which the exact function is unknown. Setting A 6-wk-old boy presented with severe hypoglycemia, unmeasurable cortisol, and grossly elevated ACTH but normal electrolytes. Genetic analysis revealed homozygote K289fs mutation in MC2R. His parents and siblings were heterozygous but phenotypically normal. Intervention and Results The role of the C terminus of MC2R was studied in two cell systems. Because the K289fs mutant changes the last eight amino acids of the protein and leads to protein elongation, wild-type MC2R and C-terminally mutated constructs were tested for activity to respond to ACTH in an OS3 cell-based reporter assay. Wild-type and alanine-substituted constructs responded normally to ACTH. By contrast K289fs and M290X had a total loss of activity. Cell surface assays and confocal localization studies revealed that K289fs and M290X receptors were not found at the cell surface, indicating that their transport from the endoplasmic reticulum to the cell membrane is disrupted. Interestingly, coimmunoprecipitation experiments showed no alteration in the interaction of mutant MC2R with MRAP, suggesting that interaction between these two proteins does not guarantee normal localization. Conclusions Loss of the C terminus of MC2R impairs cell surface expression and ACTH sensitivity but does not disrupt interaction of MC2R with MRAP. These findings highlight the extreme sensitivity of MC2R to structural disruption.
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Hirsch, Andrea, Eirini Meimaridou, Monica Fernandez-Cancio, Amit V. Pandey, María Clemente, Laura Audi, Adrian J. L. Clark, and Christa E. Flück. "Loss of the C Terminus of Melanocortin Receptor 2 (MC2R) Results in Impaired Cell Surface Expression and ACTH Insensitivity." Endocrinology 151, no. 12 (December 1, 2010): 5971. http://dx.doi.org/10.1210/endo.151.12.9989.
Анотація:
Objective: Mutations in melanocortin receptor 2 (MC2R) and its related melanocortin receptor accessory protein (MRAP) cause familial glucocorticoid deficiency. We identified a novel MC2R mutation, K289fs. This unique mutation in the C terminus of MC2R is located in the intracellular part of the protein for which the exact function is unknown. Setting: A 6-wk-old boy presented with severe hypoglycemia, unmeasurable cortisol, and grossly elevated ACTH but normal electrolytes. Genetic analysis revealed homozygote K289fs mutation in MC2R. His parents and siblings were heterozygous but phenotypically normal. Intervention and Results: The role of the C terminus of MC2R was studied in two cell systems. Because the K289fs mutant changes the last eight amino acids of the protein and leads to protein elongation, wild-type MC2R and C-terminally mutated constructs were tested for activity to respond to ACTH in an OS3 cell-based reporter assay. Wild-type and alanine-substituted constructs responded normally to ACTH. By contrast K289fs and M290X had a total loss of activity. Cell surface assays and confocal localization studies revealed that K289fs and M290X receptors were not found at the cell surface, indicating that their transport from the endoplasmic reticulum to the cell membrane is disrupted. Interestingly, coimmunoprecipitation experiments showed no alteration in the interaction of mutant MC2R with MRAP, suggesting that interaction between these two proteins does not guarantee normal localization. Conclusions: Loss of the C terminus of MC2R impairs cell surface expression and ACTH sensitivity but does not disrupt interaction of MC2R with MRAP. These findings highlight the extreme sensitivity of MC2R to structural disruption.
39
Saadi, Hadjer, Ahmed Rnnane, Rachida Touhami, and Mustapha C.E. Yagoub. "Behavioral Simulation of ISO 18000-6 Type-C Class 1 Gen2 Protocol for RFID UHF Transponder and its Application as Anti-collision Protocol in Interference Case." International Journal of Engineering & Technology 7, no. 2.28 (May 16, 2018): 276. http://dx.doi.org/10.14419/ijet.v7i2.28.12944.
Анотація:
In RFID systems, the Transponder Protocol usually uses the standard ISO 18000-6 Type-C Class 1 Generation 2, originally developed to communicate with the reader. Since a typical RFID system could be used in a myriad of tasks from product identification to environmental sensing, behavioral software functionality and hardware cost constraints are extremely constricted, principally due to their ¶standard’s requirements.¶ Thus, in this paper, an advanced behavioral simulation of the Tag ID layer of ISO 18000-6 Type-C protocol is proposed with all its states, commands and functionality, a crucial step toward effective design and test. The approach was then successfully applied to collision issues in interference case.
40
Coker, James A., Peter P. Sheridan, Jennifer Loveland-Curtze, Kevin R. Gutshall, Ann J. Auman та Jean E. Brenchley. "Biochemical Characterization of a β-Galactosidase with a Low Temperature Optimum Obtained from an Antarctic Arthrobacter Isolate". Journal of Bacteriology 185, № 18 (15 вересня 2003): 5473–82. http://dx.doi.org/10.1128/jb.185.18.5473-5482.2003.
Анотація:
ABSTRACT A psychrophilic gram-positive isolate was obtained from Antarctic Dry Valley soil. It utilized lactose, had a rod-coccus cycle, and contained lysine as the diamino acid in its cell wall. Consistent with these physiological traits, the 16S ribosomal DNA sequence showed that it was phylogenetically related to other Arthrobacter species. A gene (bgaS) encoding a family 2 β-galactosidase was cloned from this organism into an Escherichia coli host. Preliminary results showed that the enzyme was cold active (optimal activity at 18°C and 50% activity remaining at 0°C) and heat labile (inactivated within 10 min at 37°C). To enable rapid purification, vectors were constructed adding histidine residues to the BgaS enzyme and its E. coli LacZ counterpart, which was purified for comparison. The His tag additions reduced the specific activities of both β-galactosidases but did not alter the other characteristics of the enzymes. Kinetic studies using o-nitrophenyl-β-d-galactopyranoside showed that BgaS with and without a His tag had greater catalytic activity at and below 20°C than the comparable LacZ β-galactosidases. The BgaS heat lability was investigated by ultracentrifugation, where the active enzyme was a homotetramer at 4°C but dissociated into inactive monomers at 25°C. Comparisons of family 2 β-galactosidase amino acid compositions and modeling studies with the LacZ structure did not mimic suggested trends for conferring enzyme flexibility at low temperatures, consistent with the changes affecting thermal adaptation being localized and subtle. Mutation studies of the BgaS enzyme should aid our understanding of such specific, localized changes affecting enzyme thermal properties.
41
Nitzschke, A., and K. Doll. "Tetanus bei einer Färse." Tierärztliche Praxis Ausgabe G: Großtiere / Nutztiere 36, no. 02 (2008): 95–98. http://dx.doi.org/10.1055/s-0037-1621444.
Анотація:
Zusammenfassung Gegenstand und Ziel: Beschrieben werden die klinische Symptomatik, die Therapie und der Krankheitsverlauf bei einem an Tetanus erkrankten Rind. Material und Methoden: Eine 19 Monate alte Färse der Rasse Deutsche Holsteins“ wurde wegen verminderter Futteraufnahme und Pansentympanie in die Klinik eingeliefert. Die Befunde der Eingangsuntersuchung sprachen für Tetanus: steifer Gang, leicht abduzierte Gliedmaßen, steif gestellte Ohren, Vorfall des dritten Augenlids, Strabismus divergens, Pansentympanie, harte Bauchdecke. Auffälligster Laborbefund war eine metabolische Alkalose (BE +12,0 mmol/l) mit leichter Hypokaliämie (K+ 2,9 mmol/l). Eine mögliche Eintrittspforte für die Erreger war nicht erkennbar. Ergebnisse: Zur Beseitigung der Pansentympanie und zur Eingabe von Flüssigkeit und Nährstoffen wurde eine temporäre Pansenfistel angelegt. Das Tier wurde 7 Tage lang mit Procain- Penicillin behandelt (einmal täglich 50000 IE/kg KM s. c.) und erhielt zur Verminderung der Muskelspasmen Xylazin (in den ersten 10 Tagen alle 4 Stunden, danach bis zum 24. Tag alle 6 Stunden jeweils 0,11 mg/kg KM s. c.). Am 30. Tag nach Behandlungsbeginn konnte die Färse geheilt entlassen werden. Schlussfolgerung: Pansentympanie stellt bei Rindern mit Tetanus ein häufiges Symptom dar. Insofern ist diese Erkrankung differenzialdiagnostisch als Ursache einer solchen Störung mit in Betracht zu ziehen. Klinische Relevanz: Selbst bei mäßig ausgeprägter Tetanussymptomatik und erfolgreichem Ansprechen auf die Therapie muss mit einer Krankheitsdauer von etwa 4 Wochen gerechnet werden. In Anbetracht des hohen Behandlungsaufwandes kommt daher ein Therapieversuch im Wesentlichen nur bei wertvolleren Rindern infrage.
42
Franklin, Kristyn, and Anthony J. Clarke. "Overexpression and Characterization of the Chromosomal Aminoglycoside 2′-N-Acetyltransferase ofProvidencia stuartii." Antimicrobial Agents and Chemotherapy 45, no. 8 (August 1, 2001): 2238–44. http://dx.doi.org/10.1128/aac.45.8.2238-2244.2001.
Анотація:
ABSTRACT The gene coding for aminoglycoside 2′-N-acetyltransferase Ia [AAC(2′)-Ia] fromProvidencia stuartii was amplified by PCR and cloned. The resulting construct, pACKF2, was transferred intoEscherichia coli for overexpression of AAC(2′)-Ia as a fusion protein with an N-terminal hexa-His tag. The fusion protein was isolated and purified by affinity chromatography on Ni2+-nitrilotriacetic acid agarose and gel permeation chromatography on Superdex 75. Comparison of the specific activity of this enzyme with that of its enterokinase-digested derivative lacking the His tag indicated that the presence of the extra N-terminal peptide does not affect activity. The temperature and pH optima for activity of both forms of the 2′-N-acetyltransferase were 20°C and pH 6.0, respectively, while the enzymes were most stable at 15°C and pH 8.1. The Michaelis-Menten kinetic parameters for AAC(2′)-Ia at 20°C and pH 6.0 were determined using a series of aminoglycoside antibiotics possessing a 2′-amino group and a concentration of acetyl coenzyme A fixed at 10 times its K m value of 8.75 μM. Under these conditions, gentamicin was determined to be the best substrate for the enzyme in terms of bothK m andk cat/K m values, whereas neomycin was the poorest. Comparison of the kinetic parameters obtained with the different aminoglycosides indicated that their hexopyranosyl residues provided the most important binding sites for AAC(2′)-Ia activity, while the enzyme exhibits greater tolerance further from these sites. No correlation was found between these kinetic parameters and MICs determined for P. stuartiiPR50 expressing the 2′-N-acetyltransferase, suggesting that its true in vivo function is not as a resistance factor.
43
Tebbe, Jan, Peter Orth, Elke Küster-Schöck, Wolfgang Hillen, Wolfram Saenger, and Winfried Hinrichs. "Crystallization and preliminary X-ray analyses of catabolite control protein A, free and in complex with its DNA-binding site." Acta Crystallographica Section D Biological Crystallography 56, no. 1 (January 1, 2000): 67–69. http://dx.doi.org/10.1107/s0907444999013104.
Анотація:
The catabolite control protein (CcpA) from Bacillus megaterium is a member of the bacterial repressor protein family GalR/LacI. CcpA with an N-terminal His-tag was used for crystallization. Crystals of free CcpA and of CcpA in complex with the putative operator sequence (catabolite responsive elements, CRE) were obtained by vapour-diffusion techniques at 291 K using the hanging-drop method. CcpA crystals grown in the presence of polyethylene glycol 8000 belong to the hexagonal space group P6122 or P6522, with unit-cell parameters a = 74.4, c = 238.8 Å. These crystals diffract X-rays to 2.55 Å resolution and contain one monomer of the homodimeric protein per asymmetric unit. Crystals of the CcpA–CRE complex were obtained with ammonium sulfate as precipitant and belong to the tetragonal space group I4122, with unit-cell parameters a = 125, c = 400 Å and one complex per asymmetric unit. Although these co-crystals grew to a sufficient size, X-ray diffraction was limited to 8 Å resolution.
44
Gunawan, Setiyo, and Yi Hsu Ju. "Pemisahan squalene dan fatty acid sterol esters dari soybean oil deodorizer distillate." Jurnal Teknik Kimia Indonesia 7, no. 2 (October 2, 2018): 780. http://dx.doi.org/10.5614/jtki.2008.7.2.3.
Анотація:
Depending on conditions in the refining process, soybean oil deodorizer distillate (SODD) contains 42-51% free fatty acids (FFA) and 16-25% triacylglycerol (TAG). Bioactive compounds such as tocopherols, free phytosterols, fatty acid steryl esters (FASE) and squalene also make up a significant portion of SODD. The efficient removal of FFA and TAG is a crucial step in the isolation and purification of bioactive compounds from SODD. In this work, a modified soxhlet extraction technique was developed and its optimal operation conditions were determined for the efficient removal of FFA and TAG from SODD. Starting with SODD that contains 3.53% FASE and 1.99% squalene, it was possible to obtain a fraction enriched with FASE (22.0%, recovery 91.3%) and squalene (8.63%, recovery 100%) by this modified soxhlet extraction under the following operation conditions: SODD/(silica gel) = 1/3 (w/w), extraction temperature = -6°C, number of extraction/h = 8.7. FFA remained in this FASE and squalene enriched fraction is 38.7% and there was no detectable TAG. The percentage removal of FFA and TAG for this FASE and squalene enriched fraction are 84.29% and 100%, respectively. The advantages of modified soxhlet extraction over open-column chromatography are that less solvent usage, larger sample size per batch and shorter operation time.Keywords: Fatty acid steryl ester; free fatty acid; modified soxhlet extraction; soybean oil deodorizer distillate; squalene; triacylglycerol AbstrakDalam proses pemurnian minyak kedelai, produk samping dari proses penghilangan bau (soybean oil deodorizer distillate, SODD) mengandung 42-52% free fatty acids (FFA) dan 18-28% triacylglycerols (TAG). Komponen bioaktif seperti tocopherols, free phytosterols, fatty acid steryl esters (FASE) dan squalene juga mempunyai kontribusi yang besar dalam komposisi SODD. Efisiensi penghilangan FFA dan TAG adalah langkah yang sangat penting dalam pemisahan dan pemurnian komponen bioaktif dari SODD. Dalam penelitian ini, modifikasi soxhlet ekstraksi telah ditemukan dan optimisasi kondisi operasi ditentukan berdasarkan efisiensi penghilangan FFA dan TAG dari SODD. Fraksi lemak non polar (nonpolar lipid fraction, NPLF) dengan kandungan FASE (22.0%, recovery 91.3%) dan squalene (8.63%, recovery 100%) telah diperoleh dengan menggunakan modifikasi soxhlet ektraksi dengan kondisi operasi: SODD/(silica gel) = 1/3 (w/w), dan suhu ekstraksi = -6°C. FFA dan TAG yang tersisa di NPLF secara berurutan adalah 38.7% dan 0%. Persentase penghilangan FFA dan TAG di NPLF secara berurutan adalah 84.29% and 100%. Keuntungan menggunakan modifikasi soxhlet ekstraksi dibandingkan dengan silica gel column chromatography adalah sedikitnya pelarut yang digunakan, besarnya sampel per unit batch yang digunakan, dan pendeknya waktu operasi.Kata kunci: Free fatty acid, fatty acid steryl ester, modifikasi soxhlet ekstraksi, soybean oil deodorizer distillate, squalene, triacylglycerols
45
Kim, Chang Min, Jae Young Choi, Jong Hwan Yoon, and Hyun Ho Park. "Purification, crystallization and X-ray crystallographic analysis of human RAB11(S20V), a constitutively active GTP-binding form." Acta Crystallographica Section F Structural Biology Communications 71, no. 10 (September 23, 2015): 1247–50. http://dx.doi.org/10.1107/s2053230x15015447.
Анотація:
RAB11, a member of the Ras superfamily of small G proteins, is involved in the regulation of vesicle trafficking during endosome recycling. Substitution of Ser20 by Val20 in Rab11 [RAB11(S20V)] inhibits its GTP hydrolysis activity and produces a constitutively active GTP-binding form. In this study, the RAB11(S20V) mutant was overexpressed inEscherichia coliwith an engineered C-terminal His tag. RAB11(S20V) was then purified to homogeneity and was crystallized at 293 K. X-ray diffraction data were collected to a resolution of 2.4 Å from a crystal belonging to space groupI4, with unit-cell parametersa = 74.11,b= 74.11,c= 149.44 Å. The asymmetric unit was estimated to contain two molecules of RAB11(S20V).
46
Benoit, Stéphane, and Robert J. Maier. "Dependence of Helicobacter pylori Urease Activity on the Nickel-Sequestering Ability of the UreE Accessory Protein." Journal of Bacteriology 185, no. 16 (August 15, 2003): 4787–95. http://dx.doi.org/10.1128/jb.185.16.4787-4795.2003.
Анотація:
ABSTRACT The Helicobacter pylori ureE gene product was previously shown to be required for urease expression, but its characteristics and role have not been determined. The UreE protein has now been overexpressed in Escherichia coli, purified, and characterized, and three altered versions were expressed to address a nickel-sequestering role of UreE. Purified UreE formed a dimer in solution and was capable of binding one nickel ion per dimer. Introduction of an extra copy of ureE into the chromosome of mutants carrying mutations in the Ni maturation proteins HypA and HypB resulted in partial restoration of urease activity (up to 24% of the wild-type levels). Fusion proteins of UreE with increased ability to bind nickel were constructed by adding histidine-rich sequences (His-6 or His-10 to the C terminus and His-10 as a sandwich fusion) to the UreE protein. Each fusion protein was overexpressed in E. coli and purified, and its nickel-binding capacity and affinity were determined. Each construct was also expressed in wild-type H. pylori and in hypA and hypB mutant strains for determining in vivo urease activities. The urease activity was increased by introduction of all the engineered versions, with the greatest Ni-sequestering version (the His-6 version) also conferring the greatest urease activity on both the hypA and hypB mutants. The differences in urease activities were not due to differences in the amounts of urease peptides. Addition of His-6 to another expressed protein (triose phosphate isomerase) did not result in stimulation of urease, so urease activation is not related to the level of nonspecific protein-bound nickel. The results indicate a correlation between H. pylori urease activity and the nickel-sequestering ability of the UreE accessory protein.
47
Tang, Wei, Dongming Lan, Zexin Zhao, Shuang Li, Xiuting Li, and Yonghua Wang. "A Thermostable Monoacylglycerol Lipase from Marine Geobacillus sp. 12AMOR1: Biochemical Characterization and Mutagenesis Study." International Journal of Molecular Sciences 20, no. 3 (February 12, 2019): 780. http://dx.doi.org/10.3390/ijms20030780.
Анотація:
Lipases with unique substrate specificity are highly desired in biotechnological applications. In this study, a putative marine Geobacillus sp. monoacylglycerol lipase (GMGL) encoded gene was identified by a genomic mining strategy. The gene was expressed in Escherichia coli as a His-tag fusion protein and purified by affinity chromatography with a yield of 264 mg per liter fermentation broth. The recombinant GMGL shows the highest hydrolysis activity at 60 °C and pH 8.0, and the half-life was 60 min at 70 °C. The GMGL is active on monoacylglycerol (MAG) substrate but not diacylglycerol (DAG) or triacylglycerol (TAG), and produces MAG as the single product in the esterification reaction. Modeling structure analysis showed that the catalytic triad is formed by Ser97, Asp196 and His226, and the flexible cap region is constituted by residues from Ala120 to Thr160. A mutagenesis study on Leu142, Ile145 and Ile170 located in the substrate binding tunnel revealed that these residues were related with its substrate specificity. The kcat/Km value toward the pNP-C6 substrate in mutants Leu142Ala, Ile145Ala and Ile170Phe increased to 2.3-, 1.4- and 2.2-fold as compared to that of the wild type, respectively.
48
Huang, Ji, and Marie Fraser. "Pig GTP-specific succinyl-CoA synthetase in complex with succinate." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C1650. http://dx.doi.org/10.1107/s2053273314083491.
Анотація:
Succinyl-CoA synthetase (SCS) exists in the mitochondria of mammals as two different isoforms; one is ATP-specific and the other is GTP-specific. SCS is a heterodimer, and the two isoforms have a common α-subunit, but different β-subunits [1]. The β-subunit determines nucleotide specificity. Mutations in the α-subunit or the ATP-specific β-subunit can cause encephalomyopathy due to mitochondrial DNA depletion, along with lactic acidosis and methylmalonic aciduria (reviewed in [2]). The reaction catalyzed by SCS, succinyl-CoA+ NDP + Pi⇌succinate +CoA + NTP, is reversible, and the direction depends on the relative concentrations of substrates and products. Only after all substrate-binding sites are discovered can the catalytic mechanism of SCS be fully understood. Structures of SCS with ADP, GDP, GTP, Pi and CoA have been determined, but the succinate-binding site, or the binding site for the succinyl-portion of succinyl-CoA, is still unknown. Succinate is predicted to bind to the conserved sequence Gly-Gly-Ile-Val (327β-330β) located in a loop of the β-subunit of GTP-specific SCS. Crystals of other complexes with pig GTP-specific SCS have diffracted well, so we are crystallizing this enzyme in complex with succinate. Initially, plasmid containing the genes encoding pig GTP-specific SCS was transformed into E coli. After overproducing the desired protein with a 6-His tag on the C-terminus of the α-subunit, three different purification columns were used to obtain the GTP-SCS protein at high purity. Succinate was then co-crystallized with GTP-SCS under conditions containing polyethylene glycol 3350, magnesium formate and HEPES, pH 7.0.
49
Yue, Chonghui, Hongyan Ben, Junwen Wang, Tiantian Li, and Guoping Yu. "Ultrasonic Pretreatment in Synthesis of Caprylic-Rich Structured Lipids by Lipase-Catalyzed Acidolysis of Corn Oil in Organic System and Its Physicochemical Properties." Foods 8, no. 11 (November 11, 2019): 566. http://dx.doi.org/10.3390/foods8110566.
Анотація:
The current work was to evaluate the lipase-catalyzed acidolysis of corn oil with caprylic acid (CA) in organic system under bath ultrasonic pretreatment and to analyze the physicochemical properties of structured lipids (SLs). Under optimum conditions (Novozym 40086 lipase, 200 W ultrasound power, 10 min ultrasound pretreatment time, 12% dosage of lipase, Triacylglycerol (TAG)/Free fatty acids (FFA): 1/8, 40 °C for 6 h), a 45.55% CA incorporation was obtained (named SLs-U). The highest CA incorporation was 32.75% for conventional method at reaction time of 10 h (named SLs-N). The predominant TAG types of SLs were MLM (medium-, long- and medium-chain-type TAGs) and MLL (medium-, long- and long-chain-type TAGs). X-ray diffraction analysis revealed that both SLs-U and SLs-N present β form. Differential scanning calorimetry (DSC) analysis showed that both SLs-U and SLs-N show a lower melting and crystallization temperature than corn oil. This study suggested that bath ultrasonic pretreatment can accelerate lipase-catalyzed acidolysis synthesis of MLM structured lipids in an organic system, and two kinds of structured lipids show similar physicochemical properties.
50
Reid, Michael S., and George L. Staby. "A Brief History of 1-Methylcyclopropene." HortScience 43, no. 1 (February 2008): 83–85. http://dx.doi.org/10.21273/hortsci.43.1.83.
Анотація:
Research that led to the discovery of 1-methylcyclopropene (1-MCP) started with efforts to understand the effects of controlled atmosphere storage and continued with studies that examined the nature of the ethylene binding site. Although some researchers focused on the use of silver ion for inhibiting ethylene action, Sisler and his colleagues focused on analogs of olefins that had a similar effect. Efforts to tag the binding site using activation tagging with diazocyclopentadiene led to the discovery of the dramatic effects of cyclopropenes, which were identified as products of its photooxidation. The story is a testament to the value of fundamental research and the collegiality and unique intellectual and technical abilities of the primary inventor, Edward C. Sisler.