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1.
Verleih M Rebl A Köllner B Korytář T Anders E Wimmers K Goldammer T 《Molecular biology reports》2012,39(4):4291-4300
2.
Analysis of EF-hand-containing proteins in <Emphasis Type="Italic">Arabidopsis</Emphasis> 总被引:3,自引:0,他引:3
Background
In plants, calcium (Ca2+) has emerged as an important messenger mediating the action of many hormonal and environmental signals, including biotic and abiotic stresses. Many different signals raise cytosolic calcium concentration ([Ca2+]cyt), which in turn is thought to regulate cellular and developmental processes via Ca2+-binding proteins. Three out of the four classes of Ca2+-binding proteins in plants contain Ca2+-binding EF-hand motif(s). This motif is a conserved helix-loop-helix structure that can bind a single Ca2+ ion. To identify all EF-hand-containing proteins in Arabidopsis, we analyzed its completed genome sequence for genes encoding EF-hand-containing proteins. 相似文献3.
Yeong-Tae Kim Jonghee Oh Kyung-Hwan Kim Jae-Youl Uhm Byoung-Moo Lee 《Molecular biology reports》2010,37(2):717-727
Elicitins, extracellular proteins from Phytophthora fungi, elicit a hypersensitivity response (HR), including systemic acquired resistance, in some plants. The elicitin capsicein
(~10 kDa) was purified by FPLC from culture filtrates of P. capsici. Purified native and recombinant capsicein induced a hypersensitive response in leaves of the non-host plants Nicotiana glutinosa and Brassica rapa subsp. pekinensis. To search for candidate capsicein-interacting proteins from N. glutinosa, a yeast two-hybrid assay was used. We identified a protein interactor that is homologous to a serine/threonine kinase of
the plant receptor-like kinase (RLK) group and designated it NgRLK1. The ORF of NgRLK1 encodes a polypeptide of 832 amino acids (93,490 Da). A conserved domain analysis revealed that NgRLK1 has structural features
typical of a plant RLK. NgRLK1 was autophosphorylated, with higher activity in the presence of Mn2+ than Mg2+. 相似文献
4.
Cellobiohydrolase genes cbhI and cbhII were isolated from Trichoderma viride AS3.3711 and T. viride CICC 13038, respectively, using RT-PCR technique. The cbhI gene from T. viride AS3.3711 contains 1,542 nucleotides and encodes a 514-amino acid protein with a molecular weight of approximately 53.96 kDa.
The cbhII gene from T. viride CICC 13038 was 1,413 bp in length encoding 471 amino acid residues with a molecular weight of approximately 49.55 kDa. The
CBHI protein showed high homology with enzymes belonging to glycoside hydrolase family 7 and CBHII is a member of Glycoside
hydrolase family 6. CBHI and CBHII play a role in the conversion of cellulose to glucose by cutting the disaccharide cellobiose
from the non-reducing end of the cellulose polymer chain. The two cellobiohydrolase (CBHI, CBHII) genes were successfully
expressed in Saccharomyces cerevisiae H158. Maximal activities of transformants Sc-cbhI and Sc-cbhII were 0.03 and 0.089 units ml−1 under galactose induction, respectively. The optimal temperatures of the recombinant enzymes (CBHI, CBHII) were 60 and 70°C,
respectively. The optimal pHs of recombinant enzymes CBHI and CBHII were at pH 5.8 and 5.0, respectively. 相似文献
5.
Yu YJ Wu SC Chan HH Chen YC Chen ZY Yang MT 《Applied microbiology and biotechnology》2008,81(3):523-532
A novel microbial transglutaminase (TGase) from the cultural filtrate of Streptomyces netropsis BCRC 12429 (Sn) was purified. The specific activity of the purified TGase was 18.2 U/mg protein with an estimated molecular
mass of 38 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The TGase gene of S. netropsis was cloned and an open reading frame of 1,242 bp encoding a protein of 413 amino acids was identified. The Sn TGase was synthesized
as a precursor protein with a preproregion of 82 amino acid residues. The deduced amino acid sequence of the mature S. netropsis TGase shares 78.9–89.6% identities with TGases from Streptomyces spp. A high level of soluble Sn TGase with its N-terminal propeptide fused with thioredoxin was expressed in E. coli. A simple and efficient process was applied to convert the purified recombinant protein into an active enzyme and showed
activity equivalent to the authentic mature TGase.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
6.
7.
Complex signal transduction pathways underlie the myriad plant responses to attack by pathogens. Ca2+ is a universal second messenger in eukaryotes that modulates various signal transduction pathways through stimulus-specific
changes in its intracellular concentration. Ca2+-binding proteins such as calmodulin (CaM) detect Ca2+ signals and regulate downstream targets as part of a coordinated cellular response to a given stimulus. Here we report the
characterization of a tomato gene (APR134) encoding a CaM-related protein that is induced in disease-resistant leaves in response to attack by Pseudomonas syringae pv. tomato. We show that suppression of APR134 gene expression in tomato (Solanum lycopersicum), using virus-induced gene silencing (VIGS), compromises the plant’s immune response. We isolated APR134-like genes from Arabidopsis, termed CML42 and CML43, to investigate whether they serve a functionally similar role. Gene expression analysis revealed that CML43 is rapidly induced in disease-resistant Arabidopsis leaves following inoculation with Pseudomonas syringae pv. tomato. Overexpression of CML43 in Arabidopsis accelerated the hypersensitive response. Recombinant APR134, CML42, and CML43 proteins all bind Ca2+ in vitro. Collectively, our data support a role for CML43, and APR134 as important mediators of Ca2+-dependent signals during the plant immune response to bacterial pathogens.
This work was supported by a research grant (WAS) and postgraduate scholarships (DC, SLD) from the Natural Science and Engineering
Research Council of Canada, the National Science Foundation (IBN-0109633; GBM), and the Swedish Research Council (SKE). 相似文献
8.
M. P. Fernandes N. M. Inada M. R. Chiaratti F. F. B. Araújo F. V. Meirelles M. T. S. Correia L. C. B. B. Coelho M. J. M. Alves F. R. Gadelha A. E. Vercesi 《Journal of bioenergetics and biomembranes》2010,42(1):69-78
Incubation of T. cruzi epimastigotes with the lectin Cramoll 1,4 in Ca2+ containing medium led to agglutination and inhibition of cell proliferation. The lectin (50 μg/ml) induced plasma membrane
permeabilization followed by Ca2+ influx and mitochondrial Ca2+ accumulation, a result that resembles the classical effect of digitonin. Cramoll 1,4 stimulated (five-fold) mitochondrial
reactive oxygen species (ROS) production, significantly decreased the electrical mitochondrial membrane potential (ΔΨm) and impaired ADP phosphorylation. The rate of uncoupled respiration in epimastigotes was not affected by Cramoll 1,4 plus
Ca2+ treatment, but oligomycin-induced resting respiration was 65% higher in treated cells than in controls. Experiments using
T. cruzi mitochondrial fractions showed that, in contrast to digitonin, the lectin significantly decreased ΔΨm by a mechanism sensitive to EGTA. In agreement with the results showing plasma membrane permeabilization and impairment of
oxidative phosphorylation by the lectin, fluorescence microscopy experiments using propidium iodide revealed that Cramoll
1,4 induced epimastigotes death by necrosis. 相似文献
9.
Pandee P Summpunn P Wiyakrutta S Isarangkul D Meevootisom V 《Journal of microbiology (Seoul, Korea)》2011,49(2):257-264
A phytase gene was cloned from Neosartorya spinosa BCC 41923. The gene was 1,455 bp in size, and the mature protein contained a polypeptide of 439 amino acids. The deduced
amino acid sequence contains the consensus motif (RHGXRXP) which is conserved among phytases and acid phosphatases. Five possible
disulfide bonds and seven potential N-glycosylation sites have been predicted. The gene was expressed in Pichia pastoris KM71 as an extracellular enzyme. The purified enzyme had specific activity of 30.95 U/mg at 37°C and 38.62 U/mg at 42°C.
Molecular weight of the deglycosylated recombinant phytase, determined by SDS-PAGE, was approximately 52 kDa. The optimum
pH and temperature for activity were pH 5.5 and 50°C. The residual phytase activity remained over 80% of initial activity
after the enzyme was stored in pH 3.0 to 7.0 for 1 h, and at 60% of initial activity after heating at 90°C for 20 min. The
enzyme exhibited broad substrate specificity, with phytic acid as the most preferred substrate. Its K
m and V
max for sodium phytate were 1.39 mM and 434.78 U/mg, respectively. The enzyme was highly resistant to most metal ions tested,
including Fe2+, Fe3+, and Al3+. When incubated with pepsin at a pepsin/phytase ratio of 0.02 (U/U) at 37°C for 2 h, 92% of its initial activity was retained.
However, the enzyme was very sensitive to trypsin, as 5% of its initial activity was recovered after treating with trypsin
at a trypsin/phytase ratio of 0.01 (U/U). 相似文献
10.
Prabhu P Tiwari MK Jeya M Gunasekaran P Kim IW Lee JK 《Applied microbiology and biotechnology》2008,81(2):283-290
Based on analysis of the genome sequence of Bacillus licheniformis ATCC 14580, an isomerase-encoding gene (araA) was proposed as an l-arabinose isomerase (L-AI). The identified araA gene was cloned from B. licheniformis and overexpressed in Escherichia coli. DNA sequence analysis revealed an open reading frame of 1,422 bp, capable of encoding a polypeptide of 474 amino acid residues
with a calculated isoelectric point of pH 4.8 and a molecular mass of 53,500 Da. The gene was overexpressed in E. coli, and the protein was purified as an active soluble form using Ni–NTA chromatography. The molecular mass of the purified enzyme
was estimated to be ~53 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and 113 kDa by gel filtration chromatography,
suggesting that the enzyme is a homodimer. The enzyme required a divalent metal ion, either Mn2+or Co2+, for enzymatic activity. The enzyme had an optimal pH and temperature of 7.5 and 50°C, respectively, with a k
cat of 12,455 min−1 and a k
cat/K
m of 34 min−1 mM−1 for l-arabinose, respectively. Although L-AIs have been characterized from several other sources, B. licheniformis L-AI is distinguished from other L-AIs by its wide pH range, high substrate specificity, and catalytic efficiency for l-arabinose, making B. licheniformis L-AI the ideal choice for industrial applications, including enzymatic synthesis of l-ribulose. This work describes one of the most catalytically efficient L-AIs characterized thus far. 相似文献
11.
12.
Vibrio harveyi, pathogenic to fish, harbor a hemolysin gene vhh, the homologues of which are found in many species of the Genus Vibrio. In this study, we investigated the prevalence of vhh gene among V. harveyi isolated from Penaeus monodon hatcheries in India by polymerase chain reaction (PCR). The vhh was detected in 67 of the 70 V. harveyi isolates tested in this study using different combinations of PCR primers. A variant vhh gene detected in a minority of strains was cloned, sequenced, and the recombinant protein was expressed in Escherichia coli. The deduced amino acid sequence of the cloned gene was 86% similar to the previously reported amino acid sequences of VHH.
The results of this study suggest that though V. harveyi strains invariably harbor vhh, the sequence variants of the hemolysin gene exist that may impede their detection by PCR. 相似文献
13.
In recent years, the biotechnological use of xylanases has grown remarkably. To efficiently produce xylanase for food processing
and other industry, a codon-optimized recombinant xylanase gene from Streptomyces sp. S38 was synthesized and extracellularly expressed in Pichia pastoris under the control of AOX1 promoter. SDS-PAGE and activity assay demonstrated that the molecular mass of the recombinant xylanase was estimated to be
25 kDa, the optimum pH and optimum temperature were 5.5 and 50°C, respectively. In shake flask culture, the specific activity
of the xylanase activity was 5098.28 U/mg. The K
m
and V
max
values of recombinant xylanase were 11.0 mg/ml and 10000 μmol min−1 mg−1, respectively. In the presence of metal ions such as Ca2+, Cu2+, Cr3+ and K+, the activity of the enzyme increased. However, strong inhibition of the enzyme activity was observed in the presence of
Hg2+. This is the first report on the expression properties of a recombinant xylanase gene from the Streptomyces sp. S38 using Pichia pastoris. The attractive biochemical properties of the recombinant xylanase suggest that it may be a useful candidate for variety
of commercial applications. 相似文献
14.
Sanath Kumar Ammini Parvathi Ricardo L. Hernandez Kathleen M. Cadle Manuel F. Varela 《Archives of microbiology》2009,191(5):425-429
MurA [UDP-N-acetylglucosamine (UDP-NAG) enolpyruvyl transferase] is a key enzyme involved in bacterial cell wall peptidoglycan synthesis
and a target for the antimicrobial agent fosfomycin, a structural analog of the MurA substrate phosphoenol pyruvate. In this
study, we identified, cloned and sequenced a novel murA gene from an environmental isolate of Vibrio fischeri that is naturally resistant to fosfomycin. The fosfomycin resistance gene was isolated from a genomic DNA library of V. fischeri. An antimicrobial agent hypersensitive strain of Escherichia coli harboring murA from V. fischeri exhibited a high fosfomycin resistance phenotype, with minimum inhibitory concentration of 3,000 μg/ml. The cloned murA gene was 1,269 bp long encoding a 422 amino acid polypeptide with an estimated pI of 5.0. The deduced amino acid sequence
of the putative protein was identified as UDP-NAG enolpyruvyl transferase by homology comparison. The MurA protein with an
estimated molecular weight of 44.7 kDa was expressed in E. coli and purified by affinity chromatography. MurA of V. fischeri will be a useful target to identify potential inhibitors of fosfomycin resistance in pharmacological studies. 相似文献
15.
Schaloske RH Lusche DF Bezares-Roder K Happle K Malchow D Schlatterer C 《BMC cell biology》2005,6(1):13
Background
Stimulation of Dictyostelium discoideum with cAMP evokes an elevation of the cytosolic free Ca2+ concentration ([Ca2+]i). The [Ca2+]i-change is composed of liberation of stored Ca2+ and extracellular Ca2+-entry. The significance of the [Ca2+]i-transient for chemotaxis is under debate. Abolition of chemotactic orientation and migration by Ca2+-buffers in the cytosol indicates that a [Ca2+]i-increase is required for chemotaxis. Yet, the iplA - mutant disrupted in a gene bearing similarity to IP3-receptors of higher eukaryotes aggregates despite the absence of a cAMP-induced [Ca2+]i-transient which favours the view that [Ca2+]i-changes are insignificant for chemotaxis. 相似文献16.
Kateryna Zelena Holger Zorn Manfred Nimtz Ralf Günter Berger 《Archives of microbiology》2009,191(5):397-402
For the heterologous expression of the msp2 gene from the edible mushroom Marasmius scorodonius in Escherichia coli the cDNA encoding the extracellular Msp2 peroxidase was cloned into the pBAD III expression plasmid. Expression of the protein
with or without signal peptide was investigated in E. coli strains TOP10 and LMG194. Different PCR products were amplified for expression of the native target protein or a protein
with a signal peptide. Omitting the native stop codon and adding six His-residues resulted in a fusion protein amenable to
immune detection and purification by immobilised metal affinity chromatography. In E. coli the recombinant protein was produced in high yield as insoluble inclusion bodies. The influence of different parameters on
MsP2 refolding was investigated. Active enzyme was obtained by glutathione-mediated oxidation in a medium containing urea,
Ca2+, and hemin. 相似文献
17.
A novel alkylsulfatase gene, sdsAP, was cloned from a newly isolated bacterium Pseudomonas sp. S9. It encoded a protein of 675 amino acids with a calculated molecular mass of 74.9 kDa. The protein contained a typical
N-terminal signal peptide of 41 amino acid residues, followed by a metallo-β-lactamase like domain at the N-terminus and a
SCP-2-like domain at the C-terminus. This domain organization mode suggested that it belonged to the type III sulfatase. The
mature alkylsulfatase was overexpressed in Escherichia coli. The optimal temperature and pH of the recombinant SdsAP were 70°C and 9.0, respectively. Notably, at optimal conditions,
the purified recombinant SdsAP had a high specific activity of 23.25 μmol min−1 mg−1, a K
m (app) of 264.3 μmol, and a V
max (app) of 33.8 μmol min−1 mg−1 for SDS. Additionally, it still retained more than 90% activity after incubation at 65°C for 1 h, which was much different
from other alkylsulfatases reported. The recombinant enzyme hydrolyzed the primary alkyl sulfate such as sodium octyl sulfate
and sodium dodecyl sulfate (SDS). It was a Zn2+-containing and Ca2+ activated alkylsulfatase. This is the first report to explore the various characteristics of the heterologous recombinant
alkylsulfatase in details. These favorable properties could make SdsAP attractive to be useful in the degradation of SDS-containing
waste. 相似文献
18.
Identification of Three <Emphasis Type="Italic">Superoxide dismutase</Emphasis> Genes from a <Emphasis Type="Italic">Geobacillus</Emphasis> sp. 总被引:1,自引:0,他引:1
We report the characterization of three Superoxide dismutase (sod) genes isolated from a bacterium in the Geobacillus genus. We isolated the bacterium from high-temperature pond mud and used 16S rRNA gene sequence to confirm its identity in
the Geobacillus genus. The three genes Mn-sod, Fe/Mn-sod, and Cu/Zn-sod were cloned and analyzed. Their open reading frames are Mn-sod: 615 bp encoding a 204 amino acid protein; Fe/Mn-sod: 1,236 bp encoding a 411 amino acid protein; Cu/Zn-sod: 522 bp encoding a 173 amino acid protein. When these sod genes were expressed in Escherichia coli, only Mn-SOD was able to be purified. The activity of the purified Mn-SOD we got was about 2,730 U/mg. Studies of this Mn-SOD
showed that it was thermostable at 60°, had 70% activity at 80° after 2.5 h, and still had 30% activity at 90° after 2.5 h.
Mn-SOD activity required the ion Mn2+. Based on gel electrophoresis, we deduced that this Mn-SOD was a homotetramer. No activity was detected after the other two
genes (Fe/Mn-sod, Cu/Zn-sod) were expressed in Escherichia coli, but activities were detected when expressed in Pichia pastoris. 相似文献
19.
The extracellular lipase structural gene was isolated from cDNA of Aureobasidium pullulans HN2-3 by using SMARTTM RACE cDNA amplification kit. The gene had an open reading frame of 1245 bp long encoding a lipase. The coding region of the
gene was interrupted by only one intron (55 bp). It encodes 414 amino acid residues of a protein with a putative signal peptide
of 26 amino acids. The protein sequence deduced from the extracellular lipase structural gene contained the lipase consensus
sequence (G-X-S-X-G) and three conserved putative N-glycosylation sites. According to the phylogenetic tree of the lipases,
the lipase from A. pullulans was closely related to that from Aspergillus fumigatus (XP_750543) and Neosartorya fischeri (XP_001257768) and the identities were 50% and 52%, respectively. The mature peptide encoding cDNA was subcloned into pET-24a
(+) expression vector. The recombinant plasmid was expressed in Escherichia coli BL21(DE3). The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular
mass of about 47 kDa was found. Enzyme activity assay verified the recombinant protein as a lipase. A maximum activity of
0.96 U/mg was obtained from cellular extract of E. coli BL21(DE3) harboring pET-24a(+)LIP1. Optimal pH and temperature of the crude recombinant lipase were 8.0 and 35 °C, respectively and the crude recombinant lipase
had the highest hydrolytic activity towards peanut oil. 相似文献
20.
Jiquan Zhang Yuying Sun Fuhua Li Bingxin Huang Jianhai Xiang 《Molecular biology reports》2010,37(4):1913-1921