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排序方式: 共有390条查询结果,搜索用时 15 毫秒
21.
Malene Bech Vester-Christensen Maher Abou Hachem Henrik Naested Birte Svensson 《Protein expression and purification》2010,69(1):112-119
Heterologous production of large multidomain proteins from higher plants is often cumbersome. Barley limit dextrinase (LD), a 98 kDa multidomain starch and α-limit dextrin debranching enzyme, plays a major role in starch mobilization during seed germination and is possibly involved in starch biosynthesis by trimming of intermediate branched α-glucan structures. Highly active barley LD is obtained by secretory expression during high cell-density fermentation of Pichia pastoris. The LD encoding gene fragment without signal peptide was subcloned in-frame with the Saccharomyces cerevisiae α-factor secretion signal of the P. pastoris vector pPIC9K under control of the alcohol oxidase 1 promoter. Optimization of a fed-batch fermentation procedure enabled efficient production of LD in a 5-L bioreactor, which combined with affinity chromatography on β-cyclodextrin–Sepharose followed by Hiload Superdex 200 gel filtration yielded 34 mg homogenous LD (84% recovery). The identity of the recombinant LD was verified by N-terminal sequencing and by mass spectrometric peptide mapping. A molecular mass of 98 kDa was estimated by SDS–PAGE in excellent agreement with the theoretical value of 97419 Da. Kinetic constants of LD catalyzed pullulan hydrolysis were found to Km,app = 0.16 ± 0.02 mg/mL and kcat,app = 79 ± 10 s?1 by fitting the uncompetitive substrate inhibition Michaelis–Menten equation, which reflects significant substrate inhibition and/or transglycosylation. The resulting catalytic coefficient, kcat,app/Km,app = 488 ± 23 mL/(mg s) is 3.5-fold higher than for barley malt LD. Surface plasmon resonance analysis showed α-, β-, and γ-cyclodextrin binding to LD with Kd of 27.2, 0.70, and 34.7 μM, respectively. 相似文献
22.
A proteomic analysis was conducted to map the events during the initial stages of the interaction between the fungal pathogen Fusarium graminearum and the susceptible barley cultivar Scarlett. Quantification of fungal DNA demonstrated a sharp increase in fungal biomass in barley spikelets at 3 days after inoculation. This coincided with the appearance of discrete F. graminearum-induced proteolytic fragments of β-amylase. Based on these results, analysis of grain proteome changes prior to extensive proteolysis enabled identification of barley proteins responding early to infection by the fungus. In total, the intensity of 51 protein spots was significantly changed in F. graminearum-infected spikelets and all but one were identified. These included pathogenesis-related proteins, proteins involved in energy metabolism, secondary metabolism and protein synthesis. A single fungal protein of unknown function was identified. Quantitative real-time RT-PCR analysis of selected genes showed a correlation between high gene expression and detection of the corresponding proteins. Fungal genes encoding alkaline protease and endothiapepsin were expressed during 1-3 days after inoculation, making them candidates for generation of the observed β-amylase fragments. These fragments have potential to be developed as proteome-level markers for fungal infection that are also informative about grain protein quality. 相似文献
23.
Andersen JR Zein I Wenzel G Krützfeldt B Eder J Ouzunova M Lübberstedt T 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2007,114(2):307-319
Forage quality of maize is influenced by both the content and structure of lignin in the cell wall. Phenylalanine Ammonia-Lyase (PAL) catalyzes the first step in lignin biosynthesis in plants; the deamination of L-phenylalanine to cinnamic acid. Successive enzymatic steps lead to the formation of three monolignols, constituting the complex structure of lignin. We have cloned and sequenced a PAL genomic sequence from 32 maize inbred lines currently employed in forage maize breeding programs in Europe. Low nucleotide diversity and excessive linkage disequilibrium (LD) was identified at this PAL locus, possibly reflecting selective constrains resulting from PAL being the first enzyme in the monolignol, and other, pathways. While the association analysis was affected by extended LD and population structure, several individual polymorphisms were associated with neutral detergent fiber (not considering population structure) and a single polymorphism was associated with in vitro digestibility of organic matter (considering population structure). 相似文献
24.
Tian Y Cuneo MJ Changela A Höcker B Beese LS Hellinga HW 《Protein science : a publication of the Protein Society》2007,16(10):2240-2250
We report the design and engineering of a robust, reagentless fluorescent glucose biosensor based on the periplasmic glucose-binding protein obtained from Thermotoga maritima (tmGBP). The gene for this protein was cloned from genomic DNA and overexpressed in Escherichia coli, the identity of its cognate sugar was confirmed, ligand binding was studied, and the structure of its glucose complex was solved to 1.7 Angstrom resolution by X-ray crystallography. TmGBP is specific for glucose and exhibits high thermostability (midpoint of thermal denaturation is 119 +/- 1 degrees C and 144 +/- 2 degrees C in the absence and presence of 1 mM glucose, respectively). A series of fluorescent conjugates was constructed by coupling single, environmentally sensitive fluorophores to unique cysteines introduced by site-specific mutagenesis at positions predicted to be responsive to ligand-induced conformational changes based on the structure. These conjugates were screened to identify engineered tmGBPs that function as reagentless fluorescent glucose biosensors. The Y13C*Cy5 conjugate is bright, gives a large response to glucose over concentration ranges appropriate for in vivo monitoring of blood glucose levels (1-30 mM), and can be immobilized in an orientation-specific manner in microtiter plates to give a reversible response to glucose. The immobilized protein retains its response after long-term storage at room temperature. 相似文献
25.
Fetal and early post-natal development of the human spleen: from primordial arterial B cell lobules to a non-segmented organ 总被引:2,自引:2,他引:0
Immunohistological analysis of 31 human spleens from the 11th week of gestation to the early postnatal period suggested that
fetal organ development may be preliminarily divided into four stages. At stage 0 the organ anlage contained erythrocyte precursors,
few macrophages and almost no lymphocytes. Fetal spleens of stage I exhibited arterial vascular lobules and lymphocytes just
began colonizing the organ. At stage II, B and T lymphocytes formed periarteriolar clusters. B cell clusters predominated,
because B cells aggregated around the more peripheral branches of splenic arterioles, while T cells occupied the more centrally
located parts of the vessels. The vascular lobules of stage I and II consisted of central arterioles surrounded by B cells,
capillaries and peripheral venules. The lobular architecture slowly dissolved at late stage II when sinuses grew out from
the peripheral venules into the centre of the lobule. Interestingly, the B cell accumulations around peripheral arterioles
did not represent the precursors of follicles, but apparently persisted as periarteriolar B cell clusters in the adult splenic
red pulp, while follicles containing FDCs developed at late stage II from B cells in direct contact to T cell clusters around
larger arterial vessels. At stage III before birth the lobular architecture was no longer recognized. The chemokine CXCL13
was already present in vascular smooth muscle and adjacent stromal cells at stage I before B cells immigrated. CCL21, on the
contrary, was only demonstrated in fibroblast-like cells supporting T cell clusters from stage II onwards. 相似文献
26.
Birte A. Igelhorst Vanessa Niederkinkhaus Claudia Karus Maren D. Lange Irmgard D. Dietzel 《Philosophical transactions of the Royal Society of London. Series B, Biological sciences》2015,370(1672)
Effects of glial cells on electrical isolation and shaping of synaptic transmission between neurons have been extensively studied. Here we present evidence that the release of proteins from astrocytes as well as microglia may regulate voltage-activated Na+ currents in neurons, thereby increasing excitability and speed of transmission in neurons kept at distance from each other by specialized glial cells. As a first example, we show that basic fibroblast growth factor and neurotrophin-3, which are released from astrocytes by exposure to thyroid hormone, influence each other to enhance Na+ current density in cultured hippocampal neurons. As a second example, we show that the presence of microglia in hippocampal cultures can upregulate Na+ current density. The effect can be boosted by lipopolysaccharides, bacterial membrane-derived stimulators of microglial activation. Comparable effects are induced by the exposure of neuron-enriched hippocampal cultures to tumour necrosis factor-α, which is released from stimulated microglia. Taken together, our findings suggest that release of proteins from various types of glial cells can alter neuronal excitability over a time course of several days. This explains changes in neuronal excitability occurring in states of thyroid hormone imbalance and possibly also in seizures triggered by infectious diseases. 相似文献
27.
28.
Marie S. M?ller Malene B. Vester-Christensen Johanne M. Jensen Maher Abou Hachem Anette Henriksen Birte Svensson 《The Journal of biological chemistry》2015,290(20):12614-12629
Molecular details underlying regulation of starch mobilization in cereal seed endosperm remain unknown despite the paramount role of this process in plant growth. The structure of the complex between the starch debranching enzyme barley limit dextrinase (LD), hydrolyzing α-1,6-glucosidic linkages, and its endogenous inhibitor (LDI) was solved at 2.7 Å. The structure reveals an entirely new and unexpected binding mode of LDI as compared with previously solved complex structures of related cereal type family inhibitors (CTIs) bound to glycoside hydrolases but is structurally analogous to binding of dual specificity CTIs to proteases. Site-directed mutagenesis establishes that a hydrophobic cluster flanked by ionic interactions in the protein-protein interface is vital for the picomolar affinity of LDI to LD as assessed by analysis of binding by using surface plasmon resonance and also supported by LDI inhibition of the enzyme activity. A phylogenetic analysis identified four LDI-like proteins in cereals among the 45 sequences from monocot databases that could be classified as unique CTI sequences. The unprecedented binding mechanism shown here for LDI has likely evolved in cereals from a need for effective inhibition of debranching enzymes having characteristic open active site architecture. The findings give a mechanistic rationale for the potency of LD activity regulation and provide a molecular understanding of the debranching events associated with optimal starch mobilization and utilization during germination. This study unveils a hitherto not recognized structural basis for the features endowing diversity to CTIs. 相似文献
29.
Gisch N Buske B Heine H Lindner B Zähringer U 《Bioorganic & medicinal chemistry letters》2011,21(11):3362-3366
Muramyl di- and tri-peptides are putative activators of the innate immune system through stimulation of the NOD2 receptor. To provide tools for the clarification of the mechanism of this activation we isolated different UDP-muramyl tripeptides (Lys- and DAP-type) from bacteria and used them to synthesize biotinylated derivatives. All biotinylated compounds retained their ability to activate NOD2 in a cell-based test system and are therefore suitable for binding studies aimed at identifying the appropriate pattern recognition receptor(s). 相似文献
30.
Efficient secretory expression of functional barley limit dextrinase inhibitor by high cell-density fermentation of Pichia pastoris 总被引:1,自引:0,他引:1
Jensen JM Vester-Christensen MB Møller MS Bønsager BC Christensen HE Hachem MA Svensson B 《Protein expression and purification》2011,79(2):217-222
The limit dextrinase inhibitor (LDI) from barley seeds acts specifically on limit dextrinase (LD), an endogenous starch debranching enzyme. LDI is a 14 kDa hydrophobic protein containing four disulfide bonds and one unpaired thiol group previously found to be either glutathionylated or cysteinylated. It is a member of the so-called CM-protein family that includes α-amylase and serine protease inhibitors, which have been extremely challenging to produce recombinantly in functional form and in good yields. Here, LDI is produced in very high yields by secretory expression by Pichia pastoris applying high cell-density fermentation in a 5L fed-batch bioreactor. Thus about 200mg of LDI, which showed twofold higher inhibitory activity towards LD than LDI from barley seeds, was purified from 1L of culture supernatant by His-tag affinity chromatography and gel filtration. Electrospray ionization mass spectrometry verified the identity of the produced glutathionylated LDI-His(6). At a 1:1M ratio the recombinant LDI completely inhibited hydrolysis of pullulan catalyzed by 5-10 nM LD. LDI retained stability in the pH 2-12 range and at pH 6.5 displayed a half-life of 53 and 33 min at 90 and 93°C, respectively. The efficient heterologous production of LDI suggests secretory expression by P. pastoris to be a promising strategy to obtain other recombinant CM-proteins. 相似文献