scholarly journals Teladorsagia circumcincta 1,6 bisphosphate aldolase: molecular and biochemical characterisation, structure analysis and recognition by immune hosts

2020 ◽  
Author(s):  
S. Umair ◽  
C.L.G. Bouchet ◽  
N. Palevich ◽  
J.S. Knight ◽  
H.V. Simpson
Keyword(s):  
Recombinant Protein ◽  
Kinetic Properties ◽  
Dictyocaulus Viviparus ◽  
Teladorsagia Circumcincta ◽  
Multiple Alignments ◽  
Sds Page ◽  
Biochemical Characterisation ◽  
Optimum Ph ◽  
Fructose 1,6 Bisphosphate ◽  
Substrate Binding Sites

ABSTRACTA 1095 bp full length cDNA encoding Teladorsagia circumcincta aldolase (TciALDO) was cloned, expressed in Escherichia coli, the recombinant protein purified and its kinetic properties determined. A phylogenetic tree was constructed using helminth aldolase sequences. The predicted protein consisted of 365 amino acids and was present as a single band of about 44 kDa on SDS-PAGE. Multiple alignments of the protein sequence of TciALDO with homologues from other helminths showed that the greatest similarity (93%) to the aldolases of Haemonchus contortus and Dictyocaulus viviparus, 82-86% similarity to the other nematode sequences and 68-71% similarity to cestode and trematode enzymes. Substrate binding sites and conserved regions were identified and were completely conserved in other homologues. At 25 °C, the optimum pH for TciALDO activity was pH 7.5, the Vmax was 432 ± 23 nmoles.min−1.mg−1 protein and the apparent Km for the substrate fructose 1,6-bisphosphate was 0.24 ± 0.01 μM (mean ± SEM, n = 3). Antibodies in both serum and saliva from field-immune, but not nematode-naïve, sheep recognised recombinant TciALDO in enzyme-linked immunosorbent assays. The recognition of the recombinant protein by antibodies generated by exposure of sheep to native aldolase indicates similar antigenicity of the two proteins.

scholarly journals Teladorsagia circumcincta 1,6-Bisphosphate Aldolase: Molecular and Biochemical Characterisation, Structure Analysis and Recognition by Immune Hosts

Parasitologia ◽  
2021 ◽  
Vol 1 (1) ◽  
pp. 1-11
Author(s):  
Saleh Umair ◽  
Charlotte Bouchet ◽  
Nikola Palevich ◽  
Heather Simpson
Keyword(s):  
Binding Sites ◽  
Kinetic Properties ◽  
Dictyocaulus Viviparus ◽  
Teladorsagia Circumcincta ◽  
Multiple Alignments ◽  
Sds Page ◽  
Biochemical Characterisation ◽  
Optimum Ph ◽  
Fructose 1,6 Bisphosphate ◽  
Substrate Binding Sites

A 1095 bp full length cDNA encoding Teladorsagia circumcincta aldolase (TciALDO-1) was cloned and expressed in Escherichia coli. Recombinant TciALDO-1 was purified, and its kinetic properties determined. The predicted protein consisted of 365 amino acids, and was present as a single band of about 44 kDa on SDS-PAGE. Multiple alignments of the protein sequence of TciALDO-1 with homologues from other helminths showed the greatest similarity (93%) to the aldolases of Haemonchus contortus and Dictyocaulus viviparus, 82–86% similarity to the other nematode sequences, and 68–71% similarity to cestode and trematode enzymes. Substrate binding sites and conserved regions were identified, and were completely conserved in other homologues. At 30 °C, the optimum pH for TciALDO-1 activity was pH 7.5, the Vmax was 432 ± 23 nmol × min−1 × mg−1 protein, and the apparent Km for the substrate fructose 1,6-bisphosphate was 0.24 ± 0.01 µM (mean ± SEM, n = 3). Recombinant TciALDO-1 was recognized by antibodies in both serum and saliva from field-immune sheep in ELISA, however, that was not the case with nematode-naïve sheep. Teladorsagia circumcincta fructose 1,6-bisphosphate aldolase appears to have potential as a vaccine candidate to control this common sheep parasite.

scholarly journals Characterisation of a Teladorsagia circumcincta glutathione transferase

2020 ◽  
Author(s):  
Saleh Umair ◽  
Charlotte L.G. Bouchet ◽  
Qing Deng ◽  
Nikola Palevich ◽  
Heather V. Simpson
Keyword(s):  
Recombinant Protein ◽  
Glutathione Transferase ◽  
Kinetic Properties ◽  
3 Dimensional ◽  
Teladorsagia Circumcincta ◽  
Multiple Alignments ◽  
Novel Drugs ◽  
Sds Page ◽  
Vaccine Antigens ◽  
Substrate Binding Sites

ABSTRACTA 615 bp full length cDNA encoding a Teladorsagia circumcincta glutathione transferase (TcGST) was cloned, expressed in Escherichia coli and the recombinant protein purified and its kinetic properties determined. The predicted protein consisted of 205 amino acids and was present as a single band of about 24 kDa on SDS-PAGE. Multiple alignments of the protein sequence of TcGST with homologues from other helminths showed that the highest identity of 53-68% with haem-binding nematode proteins designated as members of the nu class of GSTs. Substrate binding sites and conserved regions were identified and were generally conserved. The predicted 3-dimensional structures of TcGST and HcGST revealed highly open binding cavities typical of this class of GST, considered to allow greater accessibility to diverse ligands compared with other classes of GST. At 25 °C, the optimum pH for TcGST activity was pH 7, the Vmax was 1535 ± 33 nmoles.min-1.mg-1 protein and the apparent Km for the substrate 1-chloro-2,4-dinitrobenzene (CDNB) was 0.22 ± 0.01 mM (mean ± SD, n = 2). Antibodies in both serum and saliva from field-immune, but not nematode-naÏve, sheep, recognised recombinant TcGST in enzyme-linked immunosorbent assays. The recognition of the recombinant protein by antibodies generated by exposure of sheep to the native enzyme indicates similar antigenicity of the two proteins. These findings could aid in the design of novel drugs and vaccine antigens for economically important parasites of livestock.

scholarly journals Evaluation of optimal conditions for arginase activity in streptozotocin induced diabetic rats

2012 ◽  
Vol 50 (No. 2) ◽  
pp. 69-76 ◽  
Author(s):  
M. Erisir ◽  
E. Ercel ◽  
S. Yilmaz ◽  
S. Ozan
Keyword(s):  
Maximal Activity ◽  
Diabetic Rats ◽  
Arginase Activity ◽  
Female Rats ◽  
Kinetic Properties ◽  
Ph Profile ◽  
Optimum Ph ◽  
Liver And Kidney ◽  
And Control ◽  
Assay Conditions

The assay conditions needed to achieve maximal activity of liver and kidney arginase in diabetic and non-diabetic rats were investigated and compared. The physicochemical and kinetic properties of liver arginase in diabetic and control rats were very similar, those of kidney arginase were significantly different. It was found that preincubation temperature (68&deg;C), preincubation period (20 min), optimum pH (10.1) of liver arginase and K<sub>m</sub> (3.2) for its substrate, L-arginine, did not change in diabetic and non-diabetic rats. As a consequence of diabetes, the optimum Mn<sup>2+</sup> concentration for liver arginase only changed from 1 to 2 mM. Although the preincubation temperature and period for activation of kidney arginase in control rats was unnecessary, they were found to be 56&ordm;C and 12 min in diabetic rats. The pH profile of arginase in kidney of diabetic rats was different from that of control rats. The K<sub>m</sub> value (6.7) of arginase for L-arginine in kidney is unchanged in diabetes whereas a marked decrease in V<sub>max</sub> was found. Optimum Mn<sup>2+</sup> concentration (2 mM) for kidney arginase was unchanged in diabetes. The activity of arginase in liver of diabetic animals was higher 1.5 to 1.7 times than that of controls. Diabetes caused an about 53% decrease of arginase activity in kidney of female rats, 26% in that of males. These findings may suggest an idea that encoded arginases by separate gene loci may be affected differently by the pathological and hormonal status.

scholarly journals Biosynthesis, purification, characterization and immobilization of laccase from Lithothelium sp.

Chemija ◽  
2020 ◽  
Vol 31 (3) ◽  
Author(s):  
Ingrida Radveikienė ◽  
Ingrida Pilotaitė ◽  
Rimgailė Dainytė ◽  
Regina Vidžiūnaitė
Keyword(s):  
Residual Activity ◽  
Catalytic Efficiency ◽  
Hydrophobic Interaction Chromatography ◽  
Metal Salts ◽  
Biotechnological Applications ◽  
Affinity Constants ◽  
Sds Page ◽  
Wide Range ◽  
Optimum Ph ◽  
Sulphate Salts

Novel fungal laccase isoenzymes (namely L95-1 and L95-2) produced by the Ascomycete Lithothelium sp. isolated from the forest soil were purified. However, only one of them was characterized, because the other isoenzyme lost its activity during purification. Extracellular L95-1 laccase was purified 30-fold using ion-exchange and hydrophobic interaction chromatography, with an overall yield of 88%. The molecular mass of purified L95-1 was estimated to be 85 kDa by SDS-PAGE analysis. L95-1 laccase was stable at temperature 4–22°C and pH 6.0–6.5. The substrate specificity of L95-1 laccase was examined with various compounds. Determined affinity constants (KM) varied in a wide range of 3.7–2020.0 µM, whereas catalytic efficiency constants (kcat/KM) covered a range of 0.008–1.9 µM–1 s–1. The optimum pH for most substrates varied in a range from pH 5.0 to 6.0. Sodium azide and fluoride strongly inhibited L95-1 activity, whereas sulphate salts inhibited weakly. The laccase was immobilized on the Fe3O4 nanoparticles and characterized. Residual activity remained at 20% after ten cycles of ABTS oxidation reaction. The immobilized laccase showed higher tolerance to various metal salts. The properties of L95-1 laccase make it potentially useful in the biotechnological applications.

scholarly journals Recombinant protein production provoked accumulation of ATP, fructose-1,6-bisphosphate and pyruvate in E. coli K12 strain TG1

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Jan Weber ◽  
Zhaopeng Li ◽  
Ursula Rinas
Keyword(s):  
Recombinant Protein ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Expression System ◽  
Limiting Factors ◽  
Expression Vectors ◽  
Metabolic Enzyme ◽  
E Coli ◽  
Metabolic Burden ◽  
Fructose 1,6 Bisphosphate

Abstract Background Recently it was shown that production of recombinant proteins in E. coli BL21(DE3) using pET based expression vectors leads to metabolic stress comparable to a carbon overfeeding response. Opposite to original expectations generation of energy as well as catabolic provision of precursor metabolites were excluded as limiting factors for growth and protein production. On the contrary, accumulation of ATP and precursor metabolites revealed their ample formation but insufficient withdrawal as a result of protein production mediated constraints in anabolic pathways. Thus, not limitation but excess of energy and precursor metabolites were identified as being connected to the protein production associated metabolic burden. Results Here we show that the protein production associated accumulation of energy and catabolic precursor metabolites is not unique to E. coli BL21(DE3) but also occurs in E. coli K12. Most notably, it was demonstrated that the IPTG-induced production of hFGF-2 using a tac-promoter based expression vector in the E. coli K12 strain TG1 was leading to persistent accumulation of key regulatory molecules such as ATP, fructose-1,6-bisphosphate and pyruvate. Conclusions Excessive energy generation, respectively, accumulation of ATP during recombinant protein production is not unique to the BL21(DE3)/T7 promoter based expression system but also observed in the E. coli K12 strain TG1 using another promoter/vector combination. These findings confirm that energy is not a limiting factor for recombinant protein production. Moreover, the data also show that an accelerated glycolytic pathway flux aggravates the protein production associated “metabolic burden”. Under conditions of compromised anabolic capacities cells are not able to reorganize their metabolic enzyme repertoire as required for reduced carbon processing.

scholarly journals Characterization of Biosynthetic Enzymes for Ectoine as a Compatible Solute in a Moderately Halophilic Eubacterium, Halomonas elongata

1999 ◽  
Vol 181 (1) ◽  
pp. 91-99 ◽  
Author(s):  
Hisayo Ono ◽  
Kazuhisa Sawada ◽  
Nonpanga Khunajakr ◽  
Tao Tao ◽  
Mihoko Yamamoto ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Mass ◽  
Optimum Temperature ◽  
Sds Page ◽  
Halomonas Elongata ◽  
Optimum Ph

ABSTRACT 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) is an excellent osmoprotectant. The biosynthetic pathway of ectoine from aspartic β-semialdehyde (ASA), in Halomonas elongata, was elucidated by purification and characterization of each enzyme involved. 2,4-Diaminobutyrate (DABA) aminotransferase catalyzed reversively the first step of the pathway, conversion of ASA to DABA by transamination with l-glutamate. This enzyme required pyridoxal 5′-phosphate and potassium ions for its activity and stability. The gel filtration estimated an apparent molecular mass of 260 kDa, whereas molecular mass measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 44 kDa. This enzyme exhibited an optimum pH of 8.6 and an optimum temperature of 25°C and had Km s of 9.1 mM forl-glutamate and 4.5 mM for dl-ASA. DABA acetyltransferase catalyzed acetylation of DABA to γ-N-acetyl-α,γ-diaminobutyric acid (ADABA) with acetyl coenzyme A and exhibited an optimum pH of 8.2 and an optimum temperature of 20°C in the presence of 0.4 M NaCl. The molecular mass was 45 kDa by gel filtration. Ectoine synthase catalyzed circularization of ADABA to ectoine and exhibited an optimum pH of 8.5 to 9.0 and an optimum temperature of 15°C in the presence of 0.5 M NaCl. This enzyme had an apparent molecular mass of 19 kDa by SDS-PAGE and a Km of 8.4 mM in the presence of 0.77 M NaCl. DABA acetyltransferase and ectoine synthase were stabilized in the presence of NaCl (>2 M) and DABA (100 mM) at temperatures below 30°C.

scholarly journals Purification and characterization of a thermostable ADP-glucose pyrophosphorylase from Thermus caldophilus GK-24

1996 ◽  
Vol 319 (3) ◽  
pp. 977-983 ◽  
Author(s):  
Jeong Heon KO ◽  
Cheorl Ho KIM ◽  
Dae-Sil LEE ◽  
Yu Sam KIM
Keyword(s):  
Molecular Mass ◽  
Specific Activity ◽  
Gel Filtration ◽  
Higher Plant ◽  
Sds Page ◽  
Thermus Caldophilus ◽  
Internal Peptide ◽  
Adp Glucose Pyrophosphorylase ◽  
Fructose 1,6 Bisphosphate

An extremely thermostable ADP-glucose pyrophosphorylase (AGPase) has been purified from Thermus caldophilus GK-24 to homogeneity by chromatographic methods, including gel filtration and ion-exchange and affinity chromatography. The specific activity of the enzyme was enriched 134.8-fold with a recovery of 10.5%. The purified enzyme was a single band by SDS/PAGE with a molecular mass of 52 kDa. The homotetrameric structure of the native enzyme was determined by gel filtration analysis, which showed a molecular mass of 230 kDa on a Superose-12 column, indicating that the structure of the enzyme is different from the heterotetrameric structures of higher-plant AGPases. The enzyme was most active at pH 6.0. The activity was maximal at 73–78 °C and its half-life was 30 min at 95 °C. Kinetic and regulatory properties were characterized. It was found that AGPase activity could be stimulated by a number of glycolytic intermediates. Fructose 6-phosphate, fructose 1,6-bisphosphate, phenylglyoxal and glucose 6-phosphate were effective activators, of which fructose 1,6-bisphosphate was the most effective. The enzyme was inhibited by phosphate, AMP or ADP. ATP and glucose 1-phosphate gave hyperbolic-shaped rate-concentration curves in the presence or absence of activator. A remarkable aspect of the amino acid composition was the existence of the hydrophobic and Ala+Gly residues. The N-terminal and internal peptide sequences were determined and compared with known sequences of various sources. It was apparently similar to those of AGPases from other bacterial and plant sources, suggesting that the enzymes are structurally related.

Structural features, properties and regulation of the outer-membrane protein W (OmpW) of Vibrio cholerae

Microbiology ◽  
2005 ◽  
Vol 151 (9) ◽  
pp. 2975-2986 ◽  
Author(s):  
Bisweswar Nandi ◽  
Ranjan K. Nandy ◽  
Amit Sarkar ◽  
Asoke C. Ghose
Keyword(s):  
Membrane Protein ◽  
Recombinant Protein ◽  
Outer Membrane ◽  
Vibrio Cholerae ◽  
Outer Membrane Protein ◽  
Secondary Structure Prediction ◽  
Sequence Data ◽  
Insertion Mutagenesis ◽  
Sds Page

The outer-membrane protein OmpW of Vibrio cholerae was studied with respect to its structure, functional properties and regulation of expression. On SDS-PAGE, the membrane-associated form of OmpW protein (solubilized by either 0·1 % or 2 % SDS at 25 °C) migrated as a monomer of 19 kDa that changed to 21 kDa on boiling. The protein was hyperexpressed in Escherichia coli in the histidine-tagged form and the purified His6-OmpW (heated or unheated) migrated as a 23 kDa protein on SDS-PAGE. Circular dichroism and Fourier-transform infrared spectroscopic analyses of the recombinant protein showed the presence of β-structures (∼40 %) with minor amounts (8–15 %) of α-helix. These results were consistent with those obtained by computational analysis of the sequence data of the protein using the secondary structure prediction program Jnet. The recombinant protein did not exhibit any porin-like property in a liposome-swelling assay. An antiserum to the purified protein induced a moderate level (66·6 % and 33·3 % at 1 : 50 and 1 : 100 dilutions, respectively) of passive protection against live vibrio challenge in a suckling mouse model. OmpW-deficient mutants of V. cholerae strains were generated by insertion mutagenesis. In a competitive assay in mice, the intestinal colonization activities of these mutants were found to be either only marginally diminished (for O1 strains) or 10-fold less (for an O139 strain) as compared to those of the corresponding wild-type strains. The OmpW protein was expressed in vivo as well as in vitro in liquid culture medium devoid of glucose. Interestingly, the glucose-dependent regulation of OmpW expression was less prominent in a ToxR− mutant of V. cholerae. Further, the expression of OmpW protein was found to be dependent on in vitro cultural conditions such as temperature, salinity, and availability of nutrients or oxygen. These results suggest that the modulation of OmpW expression by environmental factors may be linked to the adaptive response of the organism under stress conditions.

Cloning, expression and immunogenic characterization of theapxIIAgene ofActinobacillus pleuropneumoniae

2007 ◽  
Vol 4 (1) ◽  
pp. 63-67
Author(s):  
Yan Ke-Xia ◽  
Liu Jian-Jie ◽  
Wu Bin ◽  
Tang Xi-Biao ◽  
Cai Li-Jun ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Lethal Dose ◽  
Serum Antibody ◽  
Enzyme Linked Immunosorbent Assay ◽  
Gene Encoding ◽  
Lethal Challenge ◽  
Sds Page ◽  
Prokaryotic Expression Vector ◽  
Protection Rate ◽  
Protein Group

AbstractThe structural gene encoding ApxII toxin (apxIIA) was amplified from the genomic DNA ofActinobacillus pleuropneumoniae(APP) strain HB08 (serotype 2) and cloned into the prokaryotic expression vector pET-28a. SDS-PAGE and Western blot analysis showed that theapxIIAgene was expressed inEscherichia coliBL21 (DE3) and the expressed products could react with ApxII antibodies. The recombinant ApxIIA was purified from the inclusion bodies. Kunming mice were intraperitoneally vaccinated twice, with an interval of 2 weeks, using unfolded/refolded recombinant proteins, the native ApxII toxin extracted from the cultural supernatant of a strain of APP serotype 7 (APP-7) or phosphate-buffered saline (PBS). Serum antibody was examined by ApxIIA-specific enzyme-linked immunosorbent assay (ELISA) 2 weeks after every vaccination. Two weeks after the second vaccination, mice were challenged intraperitoneally with a lethal dose of APP-7 (1.08 × 108cfu per mouse). The protection rate reached 91.7% in the native ApxII group, 83.3% in the refolded recombinant protein group and 58.3% in the unfolded recombinant protein group, while all mice in the PBS group died within 36 h after challenge. Our data revealed that the refolded recombinant ApxIIA had excellent immunogenicity and could elicit protection against a lethal challenge of APP.

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