Cloning, Expression and Characterization of a Novel Iron Superoxide Dismutase from Magnetospirillum magneticum AMB-1

2012 ◽  
Vol 610-613 ◽  
pp. 124-128
Author(s):  
Xiang Qian Li ◽  
Qi Lei Wang ◽  
Ye Min Xue
Keyword(s):  
Superoxide Dismutase ◽  
Protein Band ◽  
Maximum Activity ◽  
Single Step ◽  
Recombinant Enzyme ◽  
Peptide Sequence ◽  
Signal Peptide Sequence ◽  
Reading Frame ◽  
Iron Superoxide Dismutase ◽  
Ph Range

The gene fesod encoding iron superoxide dismutase from Magnetospirillum AMB-1 with a calculated 22kDa was cloned and efficiently expressed in Escherichia coli BL21 (DE3). The open reading frame of 597 nucleotides encoded a protein of 199 amino acids without the signal peptide sequence. Recombinant enzyme fesod was purified by IMAC (Ni2+) in a single step to electrophoretic homogeneity presented as a single protein band on SDS-PAGE. The recombinant enzyme displayed maximum activity at 25 °C, which was stable in the pH range from 5.4 to 8.2 and at temperature from 25 to 45 °C. These results suggest that fesod may have very attractive applications in cosmetics industry as an anti ageing protein in a moderate temperature range.

scholarly journals Characterization of PauB, a Novel Broad-Spectrum Plasminogen Activator from Streptococcus uberis

2002 ◽  
Vol 184 (1) ◽  
pp. 119-125 ◽  
Author(s):  
Philip N. Ward ◽  
James A. Leigh
Keyword(s):  
Plasminogen Activator ◽  
Signal Peptide ◽  
Open Reading Frame ◽  
Peptide Sequence ◽  
Expression Cloning ◽  
Signal Peptide Sequence ◽  
Putative Gene ◽  
Reading Frame ◽  
Streptococcus Uberis ◽  
Broad Specificity

ABSTRACT A bovine plasminogen activator of atypical molecular mass (∼45 kDa) from Streptococcus uberis strain SK880 had been identified previously (L. B. Johnsen, K. Poulsen, M. Kilian, and T. E. Petersen. Infect. Immun. 67:1072–1078, 1999). The strain was isolated from a clinical case of bovine mastitis. The isolate was found not to secrete PauA, a bovine plasminogen activator expressed by the majority of S. uberis strains. Analysis of the locus normally occupied by pauA revealed an absence of the pauA open reading frame. However, an alternative open reading frame was identified within the same locus. Sequence analysis of the putative gene suggested limited but significant homology to other plasminogen activators. A candidate signal peptide sequence and cleavage site were also identified. Expression cloning of DNA encoding the predicted mature protein (lacking signal peptide) confirmed that the open reading frame encoded a plasminogen activator of the expected size, which we have named PauB. Both native and recombinant forms of PauB displayed an unexpectedly broad specificity profile for bovine, ovine, equine, caprine, porcine, rabbit, and human plasminogen. Clinical and nonclinical field isolates from nine United Kingdom sites were screened for the pauB gene and none were identified as carrying it. Similarly, clinical isolates from 20 Danish herds were all found to encode PauA and not PauB. Therefore, PauB represents a novel but rare bacterial plasminogen activator which displays very broad specificity.

Chemical and enzymatic characterization of recombinant rabbit muscle pyruvate kinase

2013 ◽  
Vol 394 (5) ◽  
pp. 695-701 ◽  
Author(s):  
Christian Boehme ◽  
Frank Bieber ◽  
Julia Linnemann ◽  
Reinhard Breitling ◽  
Stefan Lorkowski ◽  
...  
Keyword(s):  
Metal Ions ◽  
Pyruvate Kinase ◽  
Adenosine Diphosphate ◽  
Transition Metal Ions ◽  
Recombinant Enzyme ◽  
High Yield ◽  
Rabbit Muscle ◽  
Reading Frame ◽  
Ph Range ◽  
Muscle Pyruvate Kinase

Abstract The stepwise synthesis of thymidine triphosphate (TTP) requires a kinase for phosphorylation in the last step. Because pyruvate kinase (PK) using phosphoenolpyruvate (PEP) as substrate can regenerate adenosine triphosphate and phosphorylate thymidine diphosphate as well, we chose this enzyme for the synthesis of TTP via an enzymatic cascade reaction. The metalloenzyme PK shows pronounced promiscuity and therefore fits well to the conditions of this reaction. PK commonly used today is isolated from rabbit muscle. We cloned and expressed the respective open reading frame in Escherichia coli, purified, and characterized the His-tagged recombinant enzyme. The enzyme has an activity optimum at 37°C and in the pH range from 7.4 to 7.8. Km constants conformed well with the isolated native enzyme for adenosine diphosphate (ADP) to 0.37±0.02 mm and for PEP to 0.07±0.01 mm. The recombinant enzyme shows the following range in its substrate specificity: ADP>dADP>dGDP>dCDP>thymidine diphosphate (TDP). It allows the phosphorylation of TDP to TTP in high yield (up to 95%). The metal ions Mg2+ and K+ are necessary for full enzymatic activity. The addition of transition metal ions such as Mn2+, Cu2+, Co2+, and Ni2+ reduces activity. Storage of the enzyme at -20°C retains full activity.

scholarly journals Characterisation and functional analysis of canine TLR5

2020 ◽  
Vol 26 (6) ◽  
pp. 451-458
Author(s):  
Aihua Zhu ◽  
Lingling Wei ◽  
Sujuan Hu ◽  
Cheng Yang ◽  
Caifa Chen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Salmonella Enteritidis ◽  
Transmembrane Domain ◽  
Immunofluorescence Assay ◽  
Peptide Sequence ◽  
Signal Peptide Sequence ◽  
Reading Frame ◽  
Serovar Typhimurium ◽  
Receptor Domain

In this study, we characterised the single exon TLR5 gene of the Chinese rural dog. Sequence analysis revealed a 2577 nucleotide-long open reading frame of canine TLR5, encoding an 858 amino acid-long protein. The putative amino acid sequence of canine TLR5 consisted of a signal peptide sequence, 15 LRR domains, a LRR C-terminal domain, a transmembrane domain and an intracellular Toll-IL-1 receptor domain. The amino acid sequence of the canine TLR5 protein shared 95.4% identity with vulpine, 72.2% with feline and 64.7% with human TLR5. Plasmids expressing canine TLR5 and NF-κB-luciferase were constructed and transfected into HEK293T cells. Expression was confirmed by indirect immunofluorescence assay. These HEK293T cells transfected with the canine TLR5- and NF-κB-luciferase plasmids significantly responded to flagellin from Salmonella enteritidis serovar Typhimurium, indicating that it is a functional TLR5 homolog. In response to stimulation with Salmonella enteritidis, the level of TLR5 mRNA significantly increased over the control in PBMCs at 4 h. The levels of IL-8, IL-6 and IL-1β also increased after exposure. The highest levels of TLR5, IL-8 and IL-1β expression were detected at 8, 4 and 12 h after stimulation, respectively. These results imply that the expression of canine TLR5 may participate in the immune response against bacterial pathogens.

scholarly journals Purification, cloning and sequencing of an enzyme mediating the reductive dechlorination of 2,4,6-trichlorophenol from Desulfitobacterium frappieri PCP-1

2003 ◽  
Vol 373 (1) ◽  
pp. 297-303 ◽  
Author(s):  
Annie BOYER ◽  
Rachel PAGÉ-BÉLANGER ◽  
Maude SAUCIER ◽  
Richard VILLEMUR ◽  
Francois LÉPINE ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Gel Electrophoresis ◽  
Protein Band ◽  
Peptide Sequence ◽  
Ortho Position ◽  
Two Dimensional Gel Electrophoresis ◽  
Reading Frame ◽  
Reductive Dehalogenase ◽  
Apparent Homogeneity ◽  
Km Value

A new membrane-associated 2,4,6-trichlorophenol reductive dehalogenase from Desulfitobacterium frappieri PCP-1 was isolated. Initial characterization of the crude preparation showed that the dechlorinating activity was sensitive to oxygen, and its optimum pH was 7.0. Its dechlorinating activity was not inhibited by sulphate, was completely inhibited by 1 mM sulphite, and partially inhibited by 5 mM sodium azide and by more than 5 mM nitrate. Several polychlorophenols were dechlorinated in the ortho position with respect to the hydroxy group. A dehalogenase was purified to apparent homogeneity. SDS gel electrophoresis revealed a single protein band with a molecular mass of 37 kDa. However, after two-dimensional gel electrophoresis, this band was composed of three isoforms. MS analyses showed that the three isoforms were from the same protein and the molecular mass of the most abundant isoform is 33800 Da. A mixture of iodopropane and titanium citrate caused a light-reversible inhibition of the dechlorinating activity, suggesting the involvement of a corrinoid cofactor. The apparent Km value for 2,4,6-trichlorophenol and pentachlorophenol were 18.3±2.8 μM and 26.8±2.9 μM respectively, at a methyl viologen concentration of 2 mM. The N-terminal amino acid sequence and an internal tryptic peptide sequence were determined. One open reading frame (ORF) was found in the Desulfitobacterium hafniense genome containing these peptides sequences. The corresponding ORF in D. frappieri PCP-1 was cloned and sequenced. This ORF, that we designated crdA, showed no homology with any known dehalogenase, suggesting a distinct reductive dehalogenase.

Cloning, Expression, and Purification of a Cu/Zn Superoxide Dismutase from Jatropha curcas

2013 ◽  
Vol 68 (1-2) ◽  
pp. 60-69
Author(s):  
Chao Ou-yang ◽  
Shun Gao ◽  
Feng Cai ◽  
Tsair-Wang Chung ◽  
Sheng-hua Wang ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Jatropha Curcas ◽  
Metal Ion ◽  
Ethylenediaminetetraacetic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Absorption Spectrometry ◽  
Reading Frame ◽  
Flight Mass Spectrometry ◽  
Ph Range

We report cDNA cloning, expression, purification, and characterization of a novel Cu/ Zn superoxide dismutase (SOD) from Jatropha curcas leaves. The full-length cDNA of this SOD contained a 496-bp open-reading frame (ORF) encoding 162 amino acid residues. The recombinant plasmid containing the SOD coding sequence was introduced into Escherichia coli, and the SOD was expressed as a fusion protein. The recombinant SOD was purified from a high-density fed-batch culture using a combination of immobilized metal ion affinity chromatography (IMAC) and Sephadex G25 desalting chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis indicated that the recombinant SOD was a monomeric protein with a molecular mass of approximately 16.4 kDa. Isoelectric focusing showed that this SOD was a basic protein with pI values of 7.04, 7.33, 8.62, and 8.77. The activity of the SOD was stable at 70 °C for 10 min, and in a broad pH range from 4 to 9. The presence of urea (up to 8 M), guanidinium chloride (up to 6 M), and 2-mercaptoethanol (up to 8 mM) had little effect on the activity. The activity decreased gradually with increasing concentrations of imidazole, hydrogen peroxide, and ethylenediaminetetraacetic acid (EDTA). Atomic absorption spectrometry showed the presence of 0.239 copper and 0.258 zinc atoms, respectively, in the SOD polypeptide

Effect of Hubs in Amino Acid Network on Iron Superoxide Dismutase Stability

2015 ◽  
Vol 10 (2) ◽  
pp. 232-239
Author(s):  
Yanrui Ding ◽  
Xueqin Wang ◽  
Zhaolin Mou
Keyword(s):  
Superoxide Dismutase ◽  
Amino Acid ◽  
Iron Superoxide Dismutase

The iron content of iron superoxide dismutase: determination by anomalous scattering

1983 ◽  
Vol 218 (1210) ◽  
pp. 119-126 ◽  
Keyword(s):  
Superoxide Dismutase ◽  
Iron Content ◽  
Three Dimensional ◽  
Anomalous Scattering ◽  
Total Iron Content ◽  
Iron Site ◽  
Iron Superoxide Dismutase ◽  
Density Map ◽  
The Difference ◽  
Electron Density Map

The number of iron atoms in the dimeric iron-containing superoxide dismutase from Pseudomonas ovalis and their atomic positions have been determined directly from anomalous scattering measurements on crystals of the native enzyme. To resolve the long-standing question of the total amount of iron per molecule for this class of dismutase, the occupancy of each site was refined against the measured Bijvoet differences. The enzyme is a symmetrical dimer with one iron site in each subunit. The iron position is 9 ņ from the intersubunit interface. The total iron content of the dimer is 1.2±0.2 moles per mole of protein. This is divided between the subunits in the ratio 0.65:0.55; the difference between them is probably not significant. Since each subunit contains, on average, slightly more than half an iron atom we conclude that the normal state of this enzyme is two iron atoms per dimer but that some of the metal is lost during purification of the protein. Although the crystals are obviously a mixture of holo- and apo-enzymes, the 2.9 Å electron density map is uniformly clean, even at the iron site. We conclude that the three-dimensional structures of the iron-bound enzyme and the apoenzyme are identical.

scholarly journals A tetrameric iron superoxide dismutase from the eucaryote Tetrahymena pyriformis.

1990 ◽  
Vol 265 (29) ◽  
pp. 17680-17687
Author(s):  
D Barra ◽  
M E Schininà ◽  
F Bossa ◽  
K Puget ◽  
P Durosay ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Tetrahymena Pyriformis ◽  
Iron Superoxide Dismutase

scholarly journals Comprehensive analysis of the isozyme composition of laccase derived from Japanese lacquer tree, Toxicodendron vernicifluum

2021 ◽  
Vol 67 (1) ◽  
Author(s):  
Mariko Takano ◽  
Masaya Nakamura ◽  
Masanobu Tabata
Keyword(s):  
Isoelectric Focusing ◽  
Laccase Activity ◽  
Maximum Activity ◽  
Acetone Powder ◽  
Blue Color ◽  
Buffer Solution ◽  
Activity Staining ◽  
Water Extracts ◽  
Ph Range ◽  
Toxicodendron Vernicifluum

AbstractWe performed an analysis using isoelectric focusing to comprehensively clarify the isozyme composition of laccase derived from Japanese lacquer tree, Toxicodendron vernicifluum. When water extracts of acetone powder obtained from lacquer were subjected to isoelectric focusing, five bands within pI 7.35–9.30 and nine bands within pI 3.50–5.25 were detected using Coomassie staining. Similarly, laccase activity staining using guaiacol showed five bands within pI 7.35–9.30 and three bands within pI 3.50–4.25. However, laccase activity staining using gallic acid showed remarkable staining within pI 3.50–5.85, whereas staining was very weak within pI 7.35–9.30. When the water extracts of acetone powder were fractionated into the fractions containing bands within pI 7.35–9.30 and pI 3.50–5.85 by SP-Sepharose column chromatography, the former had a blue color and the latter a yellow color. The laccase activity was measured for each of the fractions in buffer solution in the pH range of 2.5–8.0. When syringaldazine, guaiacol, and 2,6-dimethoxyphenol were used as substrates, the yellow fraction showed considerably higher activity than the blue fraction for pH 5.5–7.5. When 3-methylcatechol and 4-methylcatechol were used as substrates, the yellow fraction showed higher activity for pH 4.5–6.5, and the blue fraction showed higher activity for pH 7.0–8.0. When 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) was used as the substrate, both fractions showed maximum activity at optimum pH of 3.0–4.0. Conventionally, in research on blue laccase derived from lacquer, the non-blue fraction corresponding to the yellow fraction lower than pI 6 has been removed during the purification process and thus has not been analyzed. Our results indicated that yellow laccase was present in the non-blue components of lacquer and that it may play a role in urushiol polymerization with previously reported blue laccase.

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